Selective targeting of apoptotic cells

ABSTRACT

The invention relates to a method of selectively targeting an active agent (or agent capable of becoming an active agent) to apoptotic cells in a vertebrate, comprising administering to said vertebrate a system comprising an arsenoxide (or arsenoxide equivalent) compound and said agent, wherein said system selectively targets apoptotic cells

TECHNICAL FIELD

The present Invention relates generally to targeted delivery of activeagents, wherein the active agents are generally therapeutic ordiagnostic agents. More particularly, the present invention relates to amethod of selectively targeting an active agent to apoptotic cells in avertebrate by administering a system comprising an arsenoxide (orarsenoxide equivalent) compound and at least one active agent, whereinthe arsenoxide compound selectively targets apoptotic cells and deliversthe active agent, such as therapeutic or detectable agents, to apoptoticcells and their environ.

BACKGROUND

Programmed cell death, or apoptosis, plays an integral role in cellturnover. Imbalance of apoptosis characterised by a marked increase ordecrease of apoptosis relative to cell regeneration, is often associatedwith disease (Thompson, 1995). For example, excessive apoptosis ischaracteristic of vascular disorders (Stefanec, 2000), neurodegenerativediseases (Rimon et at. 1997), myelodysplastic syndromes (Parker & Mufti,2001), ischaemia/reperfusion injury (Gottlieb & Engler, 1999), organtransplant rejection (Krams and Martinez, 1998), tumours and cancers,among others.

However, there are no specific measures of apoptosis used in patientdiagnosis or therapy. This is in part due to the lack of a convenientand sensitive marker to monitor apoptosis in vivo. Therefore, aselective marker for apoptotic cells would be advantageous in diseasediagnosis and therapy, and would also be of interest for imaging normalcell processes.

Accordingly, there is a need to selectively target active agents tosites of therapeutic or diagnostic interest in particular, there is aneed to selectively target agents to apoptotic cells.

The present invention relates to a method of targeting an active agentto apoptotic cells in a vertebrate, comprising administering to avertebrate a system comprising an arsenoxide (or arsenoxide equivalent)compound and an active agent, wherein the system selectively targetsapoptotic cells. The arsenoxide (or arsenoxide equivalent) compoundfunctions primarily as a targeting agent for delivering an active agentssuch as a therapeutic or imaging agent, to apoptotic cells with arelatively high degree of specificity. A particular advantage of thepresent invention is that the arsenoxide (or arsenoxide equivalent)compound is selectively taken up by apoptotic cells, and binds to anumber of proteins within the cell.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention there is provided amethod of selectively targeting an active agent (or agent capable ofbecoming an active agent) to apoptotic cells in a vertebrate, comprisingadministering to said vertebrate a system comprising an arsenoxide (orarsenoxide equivalent) compound and said agent wherein said systemselectively targets apoptotic cells,

Typically, the system comprises an arsenoxide (or arsenoxide equivalent)compound linked to at least one active agent, or agent capable ofbecoming an active agent

More typically, the system comprises:

(i) a first component comprising an arsenoxide (or arsenoxideequivalent) compound linked to a first binding member, and

(ii) a second component, comprising a second binding member, whereinsaid second binding member is an active agent, or an agent capable ofbecoming an active agent.

Typically, the first binding member is an enzyme and the second bindingmember is a substrate for the enzyme. Typically, the substrate for theenzyme is a pro-agent which is converted to an active agent by theenzyme. Still more typically, the enzyme substrate is a prodrug which isconverted to an active drug by the enzyme.

Even more typically, the system comprises:

(i) a first component comprising an arsenoxide (or arsenoxideequivalent) compound linked to a first binding member; and

(ii) a second component comprising a second binding member linked to atleast one active agent (or agent capable of becoming an active agent).

Typically, the first binding member is biotin and the second bindingmember is avidin or streptavidin. Still typically, the first bindingmember is avidin or streptavidin and the second binding member isbiotin.

Still typically, the first binding member is biotin, the second bindingmember is avidin or streptavidin, and the second binding member isindirectly linked to an active agent by virtue of a further bindinginteraction between the avidin or streptavidin and at least one furtherbiotin moiety which is directly linked to the active agent.

Typically, the first binding member is methotrexate and the secondbinding member is dihydrofolate reductase (DHFR). Still typically, thefirst binding member is dihydrofolate reductase and the second bindingmember is methotrexate.

Typically, the first binding member is hirudin and the second bindingmember is thrombin. Still typically, the first binding member isthrombin and the second binding member is hirudin.

Typically, the first binding member is an antigen and the second bindingmember is an antibody.

Typically, the second binding member is linked directly to an activeagent. Still typically, the second binding member is indirectly linkedto an active agent. Even more typically, indirect linking between theactive agent and the second binding member is by virtue of a furtherbinding interaction between the second binding member and at least onefurther binding member, wherein the further binding member is directlylinked to the active agent.

Yet soil more typically, the system comprises:

(i) a first component comprising an arsenoxide (or arsenoxideequivalent) compound linked to a first binding member,

(ii) a second component comprising a second binding member linked to anenzyme, and

(iii) a substrate for said enzyme.

Typically, the substrate for said enzyme is a pro-agent which isconverted to an active agent by the enzyme. Typically, the pro-agent isa prodrug.

Typically, the active agent is a therapeutic agent or a diagnosticagent.

Typically, the therapeutic agent includes agents such asradionucleotides; chemotherapeutic agents; cytotoxins; coagulants;growth factors, cytokines; bacterial, plant, or fungal endotoxins.

Typical radionucleotides suitable for use in the invention are selectedfrom the group consisting of: ³H, ¹¹C, ¹⁴C, ¹⁵O, ¹³N, ³²p, ³³p, ³⁵S,¹⁸F, ¹²⁵I, ¹²⁷I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, and ^(99m)Tc.

Typical chemotherapeutic agents include: adriamycin, taxol,fluorouricil, melphalan, cisplatin, alpha interferon, vincristine,vinblastine, angloinhibins, TNP470, pentosan polysulfate, plateletfactor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide,SP-PG and the like. Other typical chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates including busulfan; triazines including dicarbazine;ethyenimines including thiotepa and hexamethyimelamine; folic acidanalogues including methotrexate; pyrimidine analogues including5-fluorouracil, cytosine arabinoside; purine analogues including6-mercaptopurine and 6-thioguanine; antitumour antbiotics includingactinomycin D; the anthracyclines including doxorubicin, bleomycin,mitomycin C and methramycin; hormones and hormone antagonists includingtamoxifen and cortiosteroids and miscellaneous agents includingcisplatin and brequinar, and regimens such as COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine anddexamethasone), and PROMACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, taxol,etoposide/mechlorethamine. Vincristine, prednisone and procarbazine).

Typical cytotoxins include ricin.

Typical bacterial, plant, fungal endotoxins are selected from the groupconsisting of: ribosome inactivating protein, diphtheria toxin,pseudomonas endotoxin, A chain toxin, α-sarcin, aspergillin,restrictocin, and ribonucleases.

Typically, the diagnostic agent is an imaging agent More typically,substances which function as diagnostic agents are well known to thoseof ordinary skill in the art and include, fluorescent labels,radionucleotides, paramagnetic ions, X-ray imaging agents,chemiluminescent labels or labels which may be detected through asecondary enzymatic or binding step. Typical fluorescent labels suitablefor use in the system of the invention include Cy™5.5, fluorescein.Other commercial fluorescent probes suitable for use in the presentinvention are listed in the Handbook of Fluorescent Probes and ResearchProducts, 8^(th) Edition, the contents of which are incorporated hereinby reference.

Typical radionucleotides suitable for use as an imaging agent in theinvention are selected from the group consisting of:³H, ¹¹C, ¹⁴C, ¹⁵O,¹³N, ³²p, ³³p, ³⁵S, ¹⁸F, ¹²⁵I, ¹²⁷I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, and ^(99m)Tc.

Typically, paramagnetic ions are selected from the group consisting ofchromium(III), gadolinium(III), iron(II), iron (III), holmlum(III),erbium(III), manganese(II), nickel(II), copper(II), neodymium(III),yttrium(III), samarium(III), and dysprosium(III). More typically, theparamagnetic ion is gadolinium(III).

Typically, X-ray imaging agents are selected from the group consistingof gold(III), lead(II). lanthanum(III) and bismuth(III).

Typically, the active agent resides within a vehicle for delivery ofsaid agent. More typically, the vehicle For the active agent is aliposome.

Typically, the arsenoxide (or arsenoxide equivalent) compound targetsapoptotic cells. More typically, the arsenoxide (or arsenoxideequivalent) compound is of the formula (I):A-(L-Y)ρ  (I)wherein

A comprises at east one pendant group,

L comprises any suitable linker and/or spacer group;

Y comprises at least one arsenoxide or arsenoxide equivalent,

p is an integer from 1 to 10.

Typically, the sum total of carbon atoms in A and L together, is greaterthan 6.

With reference to the compound of formula (I) suitable for use in thepresent invention, typically, A is selected from the group consisting ofnatural, unnatural and synthetic amino acids, hydrophilic amines,peptides, polypeptides, oligosaccharides, and thiol containing proteins,or a combination thereof. More typically, A is selected from the groupconsisting of glutathione, glucosamine, cysteinylglycine, cysteic acid,aspartic acid, glutamic acid, lysine, and arginine, and wherein thesulfur atom of each sulfur containing compound may be optionallyoxidised to form a sulfoxide or sulfone.

Amino acid side chains are known to those of skill in the art and arelisted, for example, in standard reference texts such as King andStansfield, “A Dictionary of Genetics, 4^(th) Edition, Oxford UniversityPress, 1990, the contents of which are incorporated herein by reference.

Even more typically, A is glutathione.

Typically, p is an integer from 1to 5. Yet still more typically, p is 1.

Typically, L corresponds to (XBX′)_(n)B′. Typically, n is an integerfrom 0 to 20, more typically 0 to 15, even more typically 0 to 10, stillmore typically 0 to 5.

Still in accordance with the arsenoxide compounds suitable for use inthe present invention, the following relates to (XBX′)_(n)B′.

Typically, X is selected from the group consisting of —NR, —S(O)—,—S(O)O—, —S(O)_(2—, —S(O)) ₂O—, —C(O)—, —C(S)—, —(O)—, C(S)O—, —C(S)S—,—P(O)(R₁)—, and —P(O)(R₁)O—, or is absent;

B is selected from the group consisting of C₁-C₁₀ alkylene, C₂-C₁₀alkenylene, C₂C₁₀ alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀cycloalkenylene, C₃-C₁₀ heterocycloalkylene, C₅-C₁₀ heterocycloalkylene,C₆-C₁₂ arylene, heteroarylene and C₂-C₁₀ acyl;

X′ is selected from the group consisting of —NR—, —O—, —S—, —Se—, —S—S—,S(O)—, —OS(O)—, OS(O)—, —OS(O)₂, —OS(O)₂O—, —S(O)O—, —S(O)₂—, —S(O)₂O—,—OP(O)(R₁)—, —OP(O)(R₁)O—, —OP(O)(R₁)OP(O)(R₁)O—, —C(O)—, —C(S)—,—C(O)O—, C(S)O—, —C(S)S—, —P(O)(R₁)—, —P(O)(R₁)O—, and

or is absent wherein E is O, S, Se, NR or N(R)₂ ⁺; n is 0, 1or 2; and

B′ is selected from the group consisting of C₁-C₁₀ alkylene, C₂-C₁₀ ,C₂-C₁₀ alkenylene, alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀cycloalkenylene, C₃-C₁₀ heterocycloalkylene, C₅-C₁₀ heterocycloalkylene,C₆-C₁₂ arylene, and heteroarylene or is absent; and wherein

each R is independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycoalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl, OR₂ and C₂-C₁₀ acyl;

R′ is the same as R or two R′ may be taken together with the nitrogenatoms to which they are attached to form a 5 or 6-membered saturated orunsaturated heterocyclic ring;

each R₁ is independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkenyl,C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl,C₆-C₁₂ aryl, heteroaryl, halo, OR₂ and N(R)₂;

each R₂ is independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl and —C(O)R₅;

each R₅ is independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl, C₁-C₁₀ alkoxy, C₃-C₁₀ alkenyloxy, C₃-C₁₀ alkynyloxy,C₃-C₁₀ cycloalkyloxy, C₅-C₁₀ cycloalkenyloxy, C₃-C₁₀heterocycloalkyloxy, C₅-C₁₀ heterocycloalkenyloxy, C₆-C₁₂ aryloxy,heteroaryloxy, C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio,C₃-C₁₀ cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀heterocycloalkylthio, C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio,heteroarylthio, OH, SH and N(R)₂;

wherein for each instance that B and/or B′ is arylene, the substituentsdirectly attached to the respective arylene rings (including arsenoxideor arsenoxide equivalent) may be in a para, meta- or ortho-relationship; and

wherein each alkylene, alkenylene, alkynylene, cycloalkylene,cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, arylene,heteroarylene and acyl may be independently substituted with hydrogen,halogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀hetercycloalkenyl, C₆-C₁₂ aryl, heteroaryl, cyano, cyanate, isocyanate,OR_(2a), SR₆, nitro, arsenoxide, —(O)R_(3,) —OS(O)R₃, —S(O)₂R₃,—OS(O)₂R₃, —P(O)R₄R₄, —OP(O)R₄R₄, —N(R″)₂, —NRC(O)(CH₂)_(m)Q, —C(O)R₅;

wherein R, R₁ and R₅ are as defined above; and

R_(2a) is selected from the group consisting of hydrogen, C₁-C₅ alkyl,C₂-C₅ alkenyl, C₂-C₅ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl,C₆-C₁₂aryl, —S(O)R₃, —S(O)₂R₃, —P(O)(R₄ ₂, N(R)₂and —C(O)R₅;

each R₃ is independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₁₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl,C₆-C₁₂ aryl, heteroaryl, C₁-C₁₀ alkoxy, C₃-C₁₀ alkenyloxy, C₃-C₁₀alkynyloxy, C₃-C₁₀ cycloalkyloxy, C₅-C₁₀ cycloalkenyloxy, C₃-C₁₀heterocycloalkyloxy, C₅-C₁₀ heterocycloalkenyloxy, C₆-C₁₂ aryloxy,heteroaryloxy, C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio,C₃-C₁₀ cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀heterocycloalkylthio, C₅-C₁₀ heterocycloalkenylthio. C6-₁₂ arylthio,heteroarylthio and N(R)_(2;)

each R⁴ is independently selected from the group consisting of hydrogen,C₁-C₁₀/alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl, C₁-C₁₀ alkoxy, C₃-C₁₀ alkenyloxy, C₃-C₁₀ alkynyloxy,C₃-C₁₀ cycloalkyloxy, C₅-C₁₀ cycloalkenyloxy, C₃-C₁₀heterocycloalkyloxy, C₅-C₁₀ heterocycloalkenyloxy, C₆-C₁₂ aryloxy,heteroaryloxy, C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio,C₃-C₁₀ cycloalkylthio, C₅-C₁₀ cycloalkylthio, C₃-C₁₀heterocycloalkylthio, C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂heteroarylthio, halo and N(R)₂;

R₆ is selected from the group consisting of C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio,C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, (O)R,—S(O)R₃ and —C(O)R₅,

R″ is the same as R or two R″ taken together with the N atom to whichthey are attached may form a saturated, unsaturated or aromaticheterocyclic ring system;

Q is selected from halogen and —OS(O)₂Q₁; wherein Q₁ is selected fromC₁-C₄ alkyl, C₁-C₁-C₄ perfluoroalkyl, phenyl, p-methylphenyl; and

m is 1 to 5.

More preferably an arsenoxide compound suitable for use in the presentinvention is 4(N-(S-glutathionylacetyl)amino)-phenylarsenoxide, whichcan be abbreviated to GSAO, according to Formula IV:

Typically, in the arsenoxide compounds suitable for use in the presentinvention, the arsenoxide group (—As═O) can be replaced by an arsenoxideequivalent.

An arsenoxide equivalent any dithiol reactive species that showsessentially he same affinity towards dithiols as —As═O. Typically,arsenoxide equivalent includes dithiol reactive entities, such as As,Ge, Sn and Sb species. More typically an arsenoxide equivalent can berepresented by -D(Z₁)(Z₂). Arsenoxide equivalents are expected toexhibit identical or substantially identical activity to that of thecorresponding arsenoxide.

Typically, for arsenoxide equivalents of the form -D(Z₁)(Z₂), DE willbe, for example, As, RSn, Sb, or RGe, and Z₁ and Z₂ will be labilegroups (i.e. groups easily displaced under physiological conditons), Z₁and Z₂, may be identical or different, and may either be connected orindependent from each other (bound only to the arsenic atom).

Suitable arsenoxide equivalents include the following:-D(Z₁)(Z₂),wherein Z₁ and Z₂ are selected from the group consisting of OH, C₁-C₁₀alkoxy, C₆-C₁₀ aryloxy, C₁-C₁₀ alkylthio, C₆-C₁₀ arylthio, C₁-C₁₀alkylseleno, C₆-C₁₀ arylseleno, F, Cl, Br and I;

wherein E₁=E₂=O, E₁=O and E₂=S or E₁=E₂=S; M is R′″ and R″″ areindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₆-C12 aryl, halogen, C₁-C₁₀ alkoxy, C₆-C₁₀ aryloxy, hydroxy andcarboxy; and n=1 to 10.

For arsenoxide equivalents of the form D(Z₁)(Z₂), when D is As and Z₁and Z2 are OH, the arsenoxide equivalent may be in equilibrium withpolymeric species, as depicted below.

In respect of the equilibrium depicted above, arsenic is one of manyelements whose hydroxy species exist in equilibrium with thecorresponding polymeric anhydrides. Therefore, arsenoxide compounds mayactually exist as low or medium molecular weight polymers (eg n=3 to 6).However, the dehydration reaction is reversible, and therefore solublepolymeric anhydrides are expected to behave as arsenoxide equivalents,that is, they are expected to bind to closely spaced dithiols insubstantially the same way as the monomeric —As(OH)₂ species.

wherein X₃═NH, Y₁═O; X₃Y₁O or X₃═S, Y₁═O, and R′ is selected from thegroup consisting of hydrogen, C₁-C₁₀ alkyl, C₆-C₁₂ aryl, and carboxy, oris one of the twenty amino acid side chains;

wherein X₃═Y₁═O; X₃═NH, Y₁═O; X₃═S, Y₁═NH; or X₃═S, Y₁═NH; or X₃═S,Y₁═NH and R₁₁ to R₁₄ are selected from tie group consisting of hydrogen,C₁-C₁₀ alkyl C₆-C₁₂ aryl, and CO₂H;

wherein X₃=Y₁═O, or X₃═NH, Y₁═O; and R₁₁ to R₁₄ are selected from thegroup consisting of hydrogen, C₁-C₁₀ alkyl, C₆-C₁₂ aryl, , halogen,C₁-C₁₀ alkoxy, and CO₂H.

Typically, (XBX′)B′ is as defined above.

Typically, the method according to the first embodiment of the inventioncomprises the steps of:

(a) administering a first component of the system;

(b) optionally waiting for a period of time; and

(c) administering a second component of the system.

Typically, the period of time is between about 1 hour and about 48hours, more typically between about 3 hours and about 36 hours, stillmore typically between about 6 hours and about 24 hours. Even moretypically the period of time is about 18 hours.

Still typically, the method according to the first embodiment of theinvention comprises the steps of:

(a) administering a first component of the system;

(b) optionally waiting for a period of time;

(c) administering a second component of said system;

(d) optionally waiting for a further period of time; and

(e) administering a further binding member linked to an active agent (oragent capable of becoming an active agent).

Typically, the periods of time are as defined above.

According to a second embodiment of the invention there is provided asystem for selectively targeting an active agent (or agent capable ofbecoming an active agent) to apoptotic cells in a vertebrate, saidsystem comprising a first component comprising an arsenoxide (orarsenoxide equivalent) compound linked to at least one active agent oragent capable of becoming an active agent.

According to a third embodiment of the invention there is provided asystem for selectively targeting an active agent (or agent capable ofbecoming an active agent) to apoptotic cells in a vertebrate, saidsystem comprising

a first component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member; and

a second component composing a second binding member, wherein saidsecond binding member is an active agent or an agent capable of becomingan active agent.

According to a fourth embodiment of the invention there is provided asystem for selectively targeting an active agent (or agent capable ofbecoming an active agent) to apoptotic cells in a vertebrate, saidsystem comprising

a first component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member, and

a second component comprising a second binding member linked to anactive agent (or agent capable of becoming an active agent).

Typically, the second binding member is directly linked to the agent

Still typically, the second binding member is indirectly linked to theagent. More typically, indirect linking between the agent and the secondbinding member is by virtue of a further binding interaction between thesecond binding member and at least one further binding member, whereinthe further binding member is directly linked to the agent. Still moretypically, the first binding member and the further binding member aredifferent Even more typically, the first binding member and the furtherbinding member are the same.

Typically, with respect to any one of the second to fourth embodimentsof the invention, the first component is linked to the agent via acleavable linker, More typically, with respect to the second embodimentof the invention, the first component is linked to the agent via acleavable linker.

Typically, with respect to the third or fourth embodiment of theinvention, the first and second binding members are as described abovein accordance with the first embodiment of the invention. Stilltypically, with respect to any one of the second to fourth embodimentsof the invention, the arsenoxide compound is as described above inaccordance with the first embodiment of the invention. Still typically,the active agent is as described above in accordance with the firstembodiment of the invention.

According to a fifth embodiment of the invention there is provided amethod of treatment and/or prophylaxis of a disease in a vertebrate inneed of said treatment and/or prophylaxis, said method comprisingadministering to said vertebrate a therapeutically effective amount of asystem comprising an arsenoxide (or arsenoxide equivalent) compound anda therapeutic agent, wherein said system selectively targets apoptoticcells.

With reference to the fifth embodiment of the invention, typicallyfurther therapeutic advantages may be realised through combinationregimens. More typically, the method of treatment in accordance with thefifth embodiment of the invention may be applied in conjunction withconventional therapy, such as radiotherapy, chemotherapy or surgery.

According to a sixth embodiment of the invention there is provided theuse of a system comprising an arsenoxide (or arsenoxide equivalent)compound and a therapeutic agent for the manufacture of a medicament forthe treatment and/or prophylaxis of a disease, wherein said systemselectively targets apoptotic cells.

With reference to the fifth and sixth embodiments of the invention,typically, the disease is characterised by an imbalance of apoptosis.More typically, the disease is selected from the group consisting ofangiogenesis-dependent diseases, cellular proliferative diseases,inflammatory disorders, auto-immune diseases, blood vessel diseases,thrombosis, cancer, neurodegenerative diseases, myelodysplasticsyndromes, ischaemia/reperfusion injury and organ transplant therapy

Typically, with respect to the fifth and sixth embodiments of theinvention, the system is as described above in accordance with the firstembodiment of the invention. More typically, the therapeutic agent is asdefined in accordance with the first embodiment of the invention.

According to a seventh embodiment of the invention there is provided amethod of detecting and/or imaging apoptotic cells in a vertebrate, saidmethod comprising administering to said vertebrate a biologicallyeffective amount of a system comprising an arsenoxide (or arsenoxideequivalent) compound and a diagnostic agent, wherein said systemselectively targets apoptotic cells; and detecting said diagnostic agent

According to an eighth embodiment of the invention there is provided theuse of a system comprising an arsenoxide (or arsenoxide equivalent)compound and a diagnostic agent for the manufacture of a medicament fordetecting and/or imaging apoptotic cells, wherein said systemselectively targets apoptotic cells.

Typically, with respect to the seventh and eighth embodiments of theinvention the, system is as defined in accordance with the firstembodiment of the invention. Still typically, the diagnostic agent is asdefined in accordance with the first embodiment of the invention.

Typically, the vertebrate is selected from the group consisting ofhuman, nonhuman primate, murine, bovine, ovine, equine, caprine,leporine, avian, feline and canine. More typically, the vertebrate ishuman, nonhuman primate or murine. Even more typically, the vertebrateis human.

According to a ninth embodiment of the invention there is provided atherapeutic and/or diagnostic kit for selectively targeting an activeagent (or agent capable of becoming an active agent) to apoptotic cellsin a vertebrate, said kit comprising

a first component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member, and

a second component comprising a second binding member, wherein saidsecond binding member is an active agent or agent capable of becoming anactive agent,

optionally together with a therapeutically and/or diagnosticallyacceptable carrier and/or diluent.

According to a tenth embodiment of the invention there is provided atherapeutic and/or diagnostic kit for selectively targeting an activeagent (or agent capable of becoming an active agent) to apoptotic cellsin a vertebrate, said kit comprising

a first component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member; and

a second component comprising a second binding member linked to anactive agent (or agent capable of becoming an active agent),

optionally together with a therapeutically and/or diagnosticallyacceptable carrier and/or diluent

According to an eleventh embodiment of me invention there is provided atherapeutic and/or diagnostic kit for selectively targeting an activeagent (or agent capable of becoming an active agent) to apoptotic cellsin a vertebrate, said kit comprising

a first component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member,

a second component comprising a second binding member; and

a third component comprising a third binding member linked to an activeagent (or agent capable of becoming an active agent),

optionally together with a therapeutically and/or diagnosticallyacceptable carrier and/or diluent

Typically, With respect to the ninth to eleventh embodiments of theinvention, the components are packaged separately.

Definitions

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Furthermore,variations of the word “comprising”, such as “comprise” and “comprises”,have correspondingly varied meanings.

In the context of this specification, th term “arsenoxide” refers to thegroup —As=O.

In the context of this specification, the groups written —As=O and—As(OH)₂ are to be considered synonymous.

In the context of this specification, the term “arsenoxide equivalent”refers to any dithiol reactive species that shows essentially the sameaffinity towards dithiols as —As=O or —As(OH)_(2,) and the termincludes, for example, groups comprising a transition element and anytrivalent arsenical that is either hydrolysed to —As=O or —As(OH)₂ whendissolved in an aqueous medium (such as cell culture buffers and thefluids contained in the organism being treated).

The term “arsenical” as used herein, includes any compound that containsarsenic,

The term “acyl” as used herein, includes monovalent and divalent alkyl,alkenyl, alkynyl, cycloalkyl and cycloalkenyl moieties possessing aterminal carbonyl substituent wherein attachment may occur at thehydrocarbon moiety, the carbonyl moiety or both,

The term “alkyl” as used herein, includes within its meaning monovalent,saturated, straight and branched chain hydrocarbon radicals.

The term “alkenyl” as used herein, includes within its meaning,monovalent straight and branched chain hydrocarbon radicals having atleast one double bond. The term “alkynyl” as used herein, includeswithin its meaning, monovalent, straight and branched chain hydrocarbonradicals having at least one triple bond.

The term “alkylene” as used herein, includes within its meaningdivalent, saturated, straight chain hydrocarbon radicals.

The term “alkenylene”as used herein, includes within its meaning,divalent, straight chain hydrocarbon radicals having at least one doublebond.

The term “alkynylene” as used herein, includes within its meaning,divalent, straight chain hydrocarbon radicals having at least one triplebond.

The term “aryl” as used herein, includes within its meaning monovalent,single, polynuclear, conjugated and fused aromatic hydrocarbon radicals.

The term “arylene” as used herein, includes within its meaning divalent,single, polynuclear, conjugated and used aromatic hydrocarbon radicals.

The term “closely spaced dithiol” as used herein, includes within itsmeaning thiols that are chemically vicinal, as well as thiols broughtinto spacial apposition by virtue of molecular conformation.

The term “cycloalkyl” as used herein, includes within its meaningmonovalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic hydrocarbon radicals.

The term “cycloalkylene” as used herein, includes within its meaningdivalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic hydrocarbon radicals.

The term “cycloalkenyl” as used herein, includes within its meaningmonovalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic hydrocarbon radicals having at least one double bond.

The term cycloalkenylene” as/used herein, includes within its meaningdivalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic hydrocarbon radicals having at least one double bond.

The term “halo” as used herein, includes fluoro, chloro, bromo and iodo.

The term “heteroaryl” as used herein, includes within its meaningmonovalent, single, polynuclear, conjugated and fused aromatic radicalshaving 1 to 12 atoms wherein 1 to 6 atoms are heteroatoms selected fromO, N and S.

The term “heteroarylene” as used herein, includes within its meaningdivalent, single, polynuclear, conjugated and fused aromatic radicalshaving 1 to 12 atoms wherein 1 to 6 atoms are heteroatoms selected fromO, N and S.

The term “heterocyloalkyl” as used herein, includes within its meaningmonovalent, saturated, monocyclic, bicyclic, polycyclic or fusedradicals wherein 1 to 5 atoms are heteroatoms selected from O, N or S.

The term “heterocycloalkylene” as used herein, includes within itsmeaning divalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic radicals wherein 1 to 5 atoms are heteroatoms selected fromO, N or S.

The term “hetercycloalkenyl” as used herein, includes within its meaningmonovalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic radicals having at least 1 double bond and wherein 1 to 5atoms are heteroatoms selected from O, N or S.

The term “heterocycloalkenylene” as used herein, includes within itsmeaning divalent, saturated, monocyclic, bicyclic, polycyclic or fusedpolycyclic radicals having at least one double bond and wherein 1 to 5atoms are heteroatoms selected from O, N or S.

The term “phenylarsonic acid” as used herein, is to be consideredsynonymous with “benzene arsonic acid”.

The term “therapeutically effective amounts” as used herein, includeswithin its meaning a non-toxic but sufficient amount of a system orcomponent of a system of the invention to provide the desiredtherapeutic effect. The exact amount required will vary from subject tosubject depending on factors such as the species being treated, the ageand general condition of the subject the severity of the condition beingtreated, the particular agent being administered and the mode ofadministration and so forth. Thus, it is not possible to specify anexact “effective amount”. However, for any given case, an appropriate“effective amount” may be determined by one of ordinary skill in the artusing only routine experimentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Synthesis of GSAO. Schematic representation of the synthesis ofGSAO showing the stereochemistry and the numbering scheme used in thediscussion of the 2D ¹H—¹³C HMBC NMR spectrum.

FIG. 2. Assignment of the Structure of GSAO. An expansion of the ¹H—¹³CHMBC spectrum of GSAO in DCl/D₂O, showing the aliphatic region. Thespectrum shows any long-range heteronuclear (¹H—¹³C) coupling ascrosspeaks, in line with the corresponding ¹H and ¹³C signals along thehorizontal and vertical axes. The boxed crosspeaks correspond to ¹H—¹³Ccoupling between the C7 and C11 methylenes, confirming that alkylationby BRAO has occurred on the glutathione sulfur atom. Peaks andcrosspeaks marked “i” are due to impurites; one-bond crosspeakscorresponding to the C9 methylene and the C2 methine are also observableas doublets due to incomplete filtering by the HMBC pulse sequence.

FIG. 3: Schematic representation of the synthesis of GSAO-A.

FIG. 4. Schematic representation of the synthesis of GSAO-B.

FIG. 5: Schematic representation of the synthesis of GSAO-F.

FIG. 6: Schematic representation of the synthesis of GSAO-Cy™5.5

FIG. 7. Interaction of GSAO-B with PDI and thioredoxin. A Structure ofGSAO-B. B Purified human recombinant PDI (5 μM), human recombinantthioredoxin (5 μM) or bovine serum albumin (5 μM) was incubated withdithiothreitol (10 μM) for 60 minutes at room temperature to ensure thatthe active site disulfide(s) of PDI and thioredoxin were in the reduceddithiol form. GSAO-B (100 μM) or GSAO-B and DMP (400 μM was then addedand the reactions incubated for 30 minutes at mom temperature. Thelabelled PDI (lanes 1 and 2), thioredoxin lanes 3 and 4) and albumin(lane 5) (75 pmoles) was resolved on 4-16% SDS-PAGE, transferred to PVDFmembrane, and blotted with streptavidin-peroxidase to detect the GSAO-Blabel. The positions of M_(r) markers are shown at left.

FIG. 8. GSAO-B labels the occasional cultured cell. Confluent BAE cellswere stained with GSAO-B and visualised with streptavidin-Alexa 488, Theoccasional cell stained very brightly (see arrowhead). The vast majorityof the cells bound very little GSAO-B.

FIG. 9. GSAO labelled apoptotic cells following a caspase activation.HT1080 cells were left untreated (parts a and d) or treated withcamptothecin to induce apoptosis (parts b and e), then detached andlabelled with annexin V-PE and GSAO-F (parts a and b) or annexin V-PEand GSAA-F (parts b and e). Phycoerythrin fluorescence is plottedagainst fluorescein fluorescence. The percentage of the total cells thatlabel brightly with annexin V (UL quadrant), GSAO or GSAA (LR quadrant),or both annexin V and GSAO or GSAA (UR quadrant) is shown in parts c(GSAO) and f (GSAA). In part g, HT1080 cells were left untreated (openbars) or treated with camptothecin to induce apoptosis (closed bars),then detached and labelled with GSAO-F, annexin V-PE and propidiumiodide. Annexin V-PE fluorescence was plotted against GSAO-Ffluorescence for all cells, or all cells excluding those that hadtaken-up propidium iodide (>200 fluorescence units in FL4). In part h,HT1080 cells were treated with camptothecin in the absence or presenceof the broad-spectrum caspase inhibitor Z-VADFMK then detached andlabelled with GSAO-F. The mean fluorescence of the untreated populationwas normalised in two experiments and the bars and errors represent themean and range.

FIG. 10 GSAO entered apoptotic cells and labelled proteins containingclosely-spaced dithiols. a HT1080 cells were treated with camptothecinto induce apoptosis, then detached and labelled with GSAO-F and annexinV-Alexa-594. Cells were imaged by confocal micrscopy. Six transversesections (i to vi are shown for an annexin V-Alexa-594 labeled cell.Panel vii shows a cell that was not strongly labelled by annexinV-Alexa-594. b HT1080 cells were treated with camptothecin to induceapoptosis, then detached and incubated with annexin V-PE and GSAO-B.Cells were sorted into annexin V-positive (lane 1) and annexinV-negative (lane 2) populations. Equal numbers of cells from eachpopulation were lysed, resolved on SDS-PAGE and blotted for GSAO-B withstreptavidin-peroxidase. The positions of M_(r) markers are indicated atleft, c GSAO-B labelled proteins from part b were collected anstreptavidin-dynabeads, resolved on SDS-PAGE and Western blotted forPDI. d HT1080 cells were untreated (lanes 1 and 2) or treated (lanes 3and 4) with camptothecin, then detached and incubated with GSAO-B in theabsence (lanes 1 and 3) or presence (lanes 2 and 4) ofdimercaptopropanol. Equal numbers of cells were lysed, resolved on SDSPAGE and blotted for GSAO-B with streptavidin-peroxidase.

FIG. 11. GSAO labelled apoptotic cells in vivo and can be used to imagetumours. a Mice bearing S.C. BxPC-3 tumours in the proximal dorsum wereadministered GSAO-B or GSAA-B by S.C. injection in the hind flank Thetumours were excised after 6 hours, sectioned and stained forbiotin-labelled compound. Low power (i) and high power (ii) miorographsof a sectioned tumour show incorporation of GSAO8 into selected cells(brown). b Tumour sections described in part a were stained foractivated caspase-3 (red) and GSAO-B or GSAA-B (green) andcounterstained with a nucleic acid stain (blue). Red, green and overlayimages are shown, c Fluorescent images of a murine Lewis lung tumourgrown S.C. in a C57BL/6 mouse 1 and 24 hours after administration ofGSAO-Cy™5.5 Panel A: white light image of the Lewis lung tumour. PanelB: tumour and dorsal skin 1 hour after injection of GSAO-Cy™5.5. PanelsC and D: dorsal skin and tumour 24 hours after injection, respectivelyof Human BxPC-3 pancreatic tumours grown S. C. in SCID mice 24 hoursafter administration of GSAO-Cy™5.5, GSAA-Cy™5.5 or Cy™5.5. Panel A:white light image of a BxPC3 tumour. Panels B, C and D: tumours afterinjection of GSAA-Cy™5.5 , GSAO-Cy™5.5 or Cy™5.5, respectively.Spontaneous prostate tumour in a TRAMP mouse 24 hours afteradministration of GSAO-Cy™5.5 Panel A: white light image. PanelB:fluorescent image. The arrows indicate the margins of the central veinand tumour. The bars in c, d and e represent 1 mm.

FIG. 12. Labelling of tTF with MPB. A Unlabelled (lane 1) orMPB-labelled (lane 2) Q219C tTF (5 μg) resolved on SDS-PAGE and stainedwith Coomassie Brilliant Blue. B Unlabelled (lane 1) or MPB-labelled(lane 2) Q219C tTF (50 ng) resolved on SDS-PAGE, transferred topolyvinyldiethylene fluoride and blotted with avidin-peroxidase todetect the biotin label. The positions of M_(r) markers are indicated atleft.

FIG. 13. Formation of the GSAO-B-avidin-tTF-B complex. PDI wasimmobilised in microlitre wells, labelled with GSAO-B and incubated withtTF and increasing molar ratios of avidin. The bound tTF was detectedusing an anti-TF monoclonal antibody and peroxidase-conjugated secondaryantibody. The bars and errors are the mean ± SD of triplicatedeterminations.

FIG. 14. Carbon of the rationale for use of GSAO-B and avidin-tTF-B tothrombose tumour blood vessels.

FIG. 15. Thrombosis of tumour vasculature and necrosis of the tumour byS.C. administration of GSAO-B followed by I.V. administration ofavidin-tTF-B. A crosssection of an untreated and treated tumour isshown. Extensive necrosis of the centre of the treated tumour isapparent. The inset shows a thrombosed vessel in the treated tumour.

BEST MODE OF PERFORMING THE INVENTION

The present invention relates to the detection of apoptotic cells anduses an arsenoxide (or arsenoxide equivalent) compound to selectivelytarget an active agent, such as a therapeutic or diagnostic agent, toaopototic calls with a relatively high degree of specificity relative tonormal cells.

1. Preparation of Compounds

1.1 Synthesis of GSAO

As set out in International (PCT) Patent Application No. PCT/AU00/011434(WO 01/21628), the disclosure of which is incorporated herein byreference, arsenoxide or arsenoxide equivalent compounds, such as GSAOmay be prepared by methods known generally in the art and those skilledin the art would recognise that the various reagents and reactants canbe routinely modified in order to synthesise any given compound usefulin Fe invention. A person skilled in the art would also recognise thatthe invention also provides for the use of compounds in any state ofionisation, for example acid salt, zwitterionic uncharged, zwitterionicanion, dianion.

In a typical synthesis of a preferred arsenoxide compound for use in theinvention, glutathione may be reacted with4-(N-(bromoacetyl)amino)phenylarsenoxide (BRAO) under conditionsfavourable to the formation of a covalent bond between the free thiol ofglutathione and the chemical entity to which the arsenoxide is attachedto give GSAO. Reactions involving. nucleophilic attack by theglutathione thiol will, in general, require alkaline conditions,Electrophilic attack of some reactive species on the glutathione sulfuratom may be carded out, in general this would likely require acidicconditions. A synthesis of GSAO is provided in example 1(a) andrepresented schematically in FIG. 1.

1.2 Synthesis of GSAO-B.

A method of synthesis of GSAO-B, below, is provided in Example 1(c) andillustrated in FIG. 4.

wherein n=1 or 2.

1.3 Synthesis of GSAO-F

A method of synthesis of GSAO-F, is provided in Example 1(d) andillustrated in FIG. 5,

1.4 Synthesis of GSAO-Cy ™5.5

A method of synthesis of GSAO-Cy ™5.5, below, is provided in Example1(e) and illustrated in FIG. 6.

2. Systems for Targeting Active Agents to Apoptotic Cells and/or DeadCells

The present invention utilises systems comprising an arsenoxide (orarsenoxide equivalent) compound to selectively target an active agent toapoptotic cells. GSAO targets both apoptotic and dead calls. As outlinedin Example 3(d), at least seven proteins incorporated GSAO in apoptoticcells. Further, GSAO bound selectively to apoptoatc tumour cells invivo. As disclosed herein, this binding has been used to image tumoursin mice With a near-infrared fluorescent-labelled GSAO.

Exemplary systems suitable for use in the present invention are shownschematically below.

2.1 System 1:

One system suitable for use in the present invention comprises anarsenoxide (or arsenoxide equivalent) compound which is directly linkedto at least one active agent, or agent capable of becoming an activeagent Typically, the active agent is a therapeutic agent such as5-fluorouracil, adriamycin and vincristine, or a detectable agent suchas fluorophores, including fluorescein Cy™5.5, and 8-aminonaphthalene1,3,6trisulfonate (ANTS).

In accordance with this system, the linker may be cleavable such thatthe active agent is “released” in situ at the target site as illustratedbelow.

Cleavable linkers are well known to those skilled in the art andinclude, for example, enzymatically cleavable linkages such as peptides,esterase labile linkers, and the like.

Examples of a system useful for detecting and/or imaging apoptotic cellsare GSAO-F (FIG. 5) and GSAO-Cy™5.5 (FIG. 6), in which the arsenoxidecompound GSAO is covalently linked to the fluorophores fluorescein andCy™5.5 respectively. The GSAO moiety selectively targets apoptoticcells, and thereby selectively delivers the fluorophore to apoptoticcells (FIGS. 9-11).

By way of further example, GSAO-Cy™5.5 can be used for detectingapoptotic cells in vivo. In accordance with the method of the invention,GSAO-Cy™5.5 is administered to a subject. The mode of administrationwill vary according to the site to be detected. Typically, the mode ofadministration is parenteral, intravenous, subcutaneous, or oral. Afteradministration, a period of time is allowed to pass in order forresidual GSAO-Cy™5.5 to be cleared from general circulation, Typically,the period of time is between about 1 and about 48 hours, more typicallybetween about 3 hours and about 36 hours, still more typically betweenabout 6 hours and about 24 hours, Even more typically, the period oftime is about 18 hours, Detection of the Cy™5.5 fluorophore can beachieved using general techniques well known in the art and enables thelocation of apoptotic cells to be determined (FIG. 11).

When the system incorporates an imaging agent, such as a fluorescentagent or an MRI agent (e.g. a paramagnetic lanthanide ion complex ofDOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid), or DTPA(diethylenetriaminepentaacetic acid)), the imaging agent is selectivelytargeted to sites of apoptotic cells, Standard methods of detection ofthe imaging agent will then enable those sites to be readily identified.

Gd(III) is one example of a lanthanide ion which has been shown to bevery effective as a contrast-enhancing agent in magnetic resonanceimaging (MRI). The following schemes depict the synthesis of a systemcomprising an arsenoxide compound (represented by GSAO) which isdirectly linked to a lanthanide ion complex, represented by Gd(III) DOTAand Gd(III) DPTA, suitable for use in the present invention.

2.1.1 Detectable Systems Comprising Lanthanide Ions(a) Synthesis of GSAO-[Gd(III) DOTA]

(b) Synthesis of GSAO-[Gd(III) DTPA]

The linking group been the GSAO moiety and the DOTA or DTPA complexexemplified in the above schemes ran be readily prepared by thoseskilled in the art using known chemical reactions. One example of asynthetic mule is depicted below.

An alternative approach to the preparation of arsenoxide-[anthanidecomplex] conjugates utilises the functionalised DOTA and DTPA ligandssuch as the isothiocyanate derivatives shown below:

The above ligands have all been well defined for their stability withGd(III), Y(III) and other lanthanide ions. An example of a syntheticmute to arsenoxide conjugates of the above ligands is depicted in thescheme below using the CHX-A″ ligand. The arsenoxide moiety isexemplified by GSAO.

2.1.3 Detectable and/or Therapeutic Systems Comprising Radionucleotides

Radioisotopes are useful as both imaging and therapeutic agents. Asynthetic route to systems suitable for use in the present invention inwhich an arsenoxide moiety (exemplified by GSAO) is linked to aradiolabelled moiety is shown below.

The scheme shown above utilises iodine-123, however a person skilled inthe art would readily comprehend that the above synthesis is general andalso applies to other active agents and radioisotopes. For instance,substitution of sodium [^(123I)]iodide with sodium[^(125I)]iodide or[^(131I)]iodide would give a system comprising the corresponding,radio-iodine radionucleotide, As illustrated below, a ¹⁸F fluorinatedsystem can be readily prepared in a similar manner to the abovesynthesis.

¹⁸F is a known positron emitter and is used in positron emissiontomography (PET) as an imaging agent. Other radioisotopes suitable foruse in PET systems are well known in the art, e.g. ⁸⁶Y and ⁹⁹Tc.Accordingly, a further system of the invention in which the active agentdirectly linked to an arsenoxide moiety is a PET agent, is thetechnetium-99 labelled system prepared according to the scheme below.

In addition to use in MRI, arsenoxide conjugates of the ligands CHX-A″,1B4M or C-DOTA (above) may also be radiolabelled, for example, with¹¹¹in for use in γ-scintigraphy, or with ⁶⁴Cu, ¹⁸F, ⁸⁸Y or ⁹⁹Tc for usein PET imaging. Other radioisotopes useful for therapeutic purposeswithin the scope of the present invention include rhenium-188,copper-64, indium-111, lutetium-177, and yttrium-90, bismuth-213. Otherradioisotopes are well known to those skilled in the art. Thesuitability of a radioisotope for therapeutic applications can bereadily identified based on targeting kinetics and results frombiodistribution studies.

2.2 System II:

Another system suitable for use in the present invention, illustratedschematically below, comprises (i) a first component comprising anarsenoxide (or arsenoxide equivalent) compound which is linked to afirst binding member; and (ii) a second component, comprising a secondbinding member, wherein the second binding member is an active agent, oran agent capable of becoming an active agent.

The arsenoxide (or arsenoxide equivalent) moiety, such as GSAO,selectively targets apoptotic cells thereby delivering the first bindingmember to apoptotic cells. The first binding member is capable ofinteracting with a second binding member. For example, a first bindingmember is an enzyme and the second binding member is a substrate for theenzyme. The enzyme substrate may be a prodrug which is converted to anactive drug by the enzyme at the site of apoptotic cells. In particular,this system can utilise the ‘bystander effect’ whereby the active drugcan diffuse to nearby cells in the environ of the apoptotic cell(s)targeted by the above system,

In accordance with the method of the present invention, the firstcomponent of this system could be administrated to a subject followed bya period of time to allow any of the component not taken up by apoptoticcells to be cleared from general circulation. The second component, viz,the second binding member can then be administered to the subject Themode of administration is typically parenteral, subcutaneous,intravenous or oral. The second binding member will interact with thefirst binding member which is selectively located at apoptotic cells,thereby enabling the therapeutic or detectable agent to be isolated inthe apoptotic cell environment.

2.3 stem III:

A further system suitable for use in the present invention, illustratedschematically below, comprises (i) a first component comprising anarsenoxide (or arsenoxide equivalent) compound which is linked to afirst binding member, and (ii) a second component comprising a secondbinding member linked to at least one active agent.

According to this system, an arsenoxide moiety (such as GSAO) is linkedto a first binding member (such as biotin) ie, an example of a firstcomponent of this system is GSAO-B. When administered to a subject, thearsenoxide moiety selectively targets GSAO-B to apoptotic cells. Thesecond component comprises a second binding member which is capable ofinteracting with the first binding member. The second binding member maybe linked to an active agent such as a therapeutic agent (e.g. achemotherapeutic drug) or a diagnostic agent (e.g, a fluorophore). Thesecond binding member may be linked to a vehicle for the active agent,such as a liposome, and the active agent may reside within the vehicle,ie, within the liposome. When the second component is administered tothe subject, it selectively binds to the first component at the site ofapoptotic cells. In the case of GSAO-B where the first binding member isbiotin, a suitable second binding member is avidin or streptavidin.

Typically, after the first component of the system is administered to asubject this is followed by a period of time to enable any of theresidual first component not taken up by apoptotic cells to be clearedfrom circulation. The second component is then administered.

In one example in accordance with this system, the first component cancomprise GSAO-B and the second component can comprise avidin linked to aliposome which encapsulates an active agent such as a chemotherapeuticdrug, e.g. deoxyrubicin. Typically, the liposome Will be coated withavidin. This system allows high concentrations of an active agent (suchas deoxyrubicin) to be selectively targeted to apoptotic cells relativeto normal cells.

A further example of this system for use in the present invention isprovided by Examples 3(d) and 3(e). In example 3(d), cells were treatedwith the first component GSAO-B, in vitro, and residual GSAO-B clearedby washing. The second component, comprising avidin-peroidase was thenadded. A known method of detection allowed apoptotic cells to beidentified.

2.4 System IV:

Another system suitable for use in the present invention is representedschematically below.

An example of this system used in accordance with the method of theinvention is provided in Example 5(c). As disclosed herein, the propertyof GSAO targeting apoptotic tumour cells has been used to deliver aclotting agent to tumour blood vessels. Tissue Factor (TF) is the majorInitiating receptor for the blood coagulation cascade. Binding of factorVII/VIIa to TF activates the serine protelnase zymogens factors IX and Xby limited proteolysis leading to the formation of thrombin andultimately a blood clot. The extracellular domain of TF (tTF) is asoluble protein with a factor X-activating activity that is about fiveorders of magnitude less than that of native transmembrane TF in anappropriate phospholipid membrane environment. This is because theTF:VIIa complex binds and activates factors IX and X far moreefficiently when associated with a negatively charged surface.

When administered to a vertebrate having, for example, a tumour, thearsenoxide compound to delivers Tissue Factor almost exclusively to thesite of increased apoptosis, that is, the tumour site(s). Once localisedat the tumour site, the presence of Tissue Factor can initiate thethrombin cascade to induce site-specific blood clotting, therebyobstructing bloodflow to the tumour and causing severe necrosis oftumour tissue.

3. Pharmaceutical and/or Therapeutic Formulations

Further, components of the systems of the invention often involve theactive agents outlined above present in the form of pharmaceuticaland/or therapeutic formulations, that is, active agents present togetherwith a pharmaceutically acceptable carrier, adjuvant and/or diluent.

For medical use, salts of the active agents may be used in the systemsof the invention and they include pharmaceutically acceptable salts,although other salts may be used in the preparation of the compound orof the pharmaceutically acceptable salt thereof. By pharmaceuticallyacceptable salt it is meant those salts which, within the scope of soundmedical judgement, are suitable for use in contact with the tissues ofhumans and lower animals without undue toxicity, irritation, allergicresponse and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art

For instance, suitable pharmaceutically acceptable salts of thecompounds useful in the systems of the invention may be prepared bymixing a pharmaceutically acceptable acid such as hydrochloric acid,sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleicacid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonicacid, tartaric acid, or citric acid with 30 the compounds of theinvention. Suitable pharmaceutically acceptable salts of the compoundsuseful in the systems of the invention therefore include acid additionsalts.

For example, S. M. Berge et al. describe pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. The saltscan be prepared in situ during the final isolation and purification ofthe compounds used in the systems of the invention, or separately byreacting the free base function with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate,ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, phenylproplonate,phosphate, picrate, pivalate,. propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

Also included within the scope of compounds useful in the systems of thepresent invention is are prodrugs. Typically, prodrugs will befunctional derivatives of the compounds used in the present inventionwhich are readily converted in vivo to the required (active) compoundsas used in the systems of the invention as active agents, such astherapeutic and/or diagnostic agents. Typical procedures for theselection and preparation of prodrugs are known to those of skill in theart and are described, for instance, in H. Bundgaard (Ed), Design ofProdrugs,.Elsevier, 1985.

Single or multiple administrations of the compounds or pharmaceuticalcompositions can be carried out with dose levels and pattern beingselected by the treating physician. Regardless, the compounds orpharmaceutical compositions useful in systems of the present inventionshould provide a quantity of the compound sufficient to effectivelytreat the patient.

One skilled in the art would be able, by routine experimentation, todetermine an effective, non-toxic amount of the compounds orpharmaceutical compositions used in the invention which would berequired to detect apoptotic cells and/or treat or prevent the disordersand diseases. Generally, an effective dosage is expected to be in therange of about 0.0001 mg to about 1000 mg per kg body weight per 24hours; typically, about 0.001 mg to about 750 mg per kg body weight per24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours;about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1mg to about 250 mg per kg body weight per 24 hours; about 1.0 mg toabout 250 mg per kg body weight per 24 hours. More typically, aneffective dose range is expected to be in the range about 1.0 mg toabout 200 mg per kg body weight per 24 hours; about 1.0 mg to about 100mg per kg body weight per 24 hours; about 1.0 mg to about 50 mg per kgbody weight per 24 hours; about 1.0 mg to about 25 mg per kg body weightper 24 hours; about 5.0 mg to about 50 mg per kg body weight per 24hours; about 5.0 mg to about 20 mg per kg body weight per 24 hours;about 5.0 mg to about 15 mg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500 mg/m².Generally, an effective dosage is expected to be in the range of about25 to about 500 mg/m², preferably about 25 to about 350 Mg/m², morepreferably about 25 to about 300 mg/m², still more preferably about 25to about 250 mg/m², even more preferably about 50 to about 250mg/m², andstill even more preferably about 75 to about 150 Mg/m².

In relation to GSAO, an effective dosage is in the range of about 0.0001mg to about 100 mg GSAO per kg body weight per 24 hours, preferablyabout 0.001 mg to about 100 mg GSAO per kg body weight per 24 hours,more preferably about 0.01 mg to about 50 mg GSAO per kg body weight per24 hours, even more preferably about 0.1 mg to about 20 mg GSAO per kgbody weight per 24 hours, even more preferably still about 0.1 mg toabout 10 mg GSAO per 1 body weight per 24 hours.

In relation to an active agent for use in systems of the presentinvention, an effective dosage is in the range of about 0.0001 mg toabout 100 mg agent per kg body weight per 24 hours, preferably about0.001 mg to about 100 mg agent per kg body weight per 24 hours, morepreferably about 0.01 mg to about 50 mg agent per kg body weight per 24hours, even more preferably about 0.1 mg to about 20 mg agent per kgbody weight per 24 hours, even more preferably still about 0.1 mg toabout 10 mg agent per kg body weight per 24 hours.

Typically the treatment would be for the duration of the condition.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and a spacing of individual dosages of a compoundused in a system of the present invention will be determined by thenature and extent of the condition being treated, the form, route andsite of administration, and the nature of the particular vertebratebeing treated. Also, such optimum conditions can be determined byconventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thecompound within the systems of the present invention given per day for adefined number of days, can be ascertained by those skilled in the artusing conventional course of treatment determination tests.

Whilst the compounds used in the systems of the present invention may beadministered alone, it is generally preferable that the compound beadministered as a pharmaceutical composition/formulation. In generalpharmaceutical formulations representing the component(s) of the systemsof the present invention may be prepared according to methods which areknown to those of ordinary skill in the art and accordingly may includea pharmaceutically acceptable carrier, diluent and/or adjuvant.

The carriers, diluents and adjuvants must be “acceptable” in terms ofbeing compatible with the other ingredients of the formulation, and notdeleterious to the recipient thereof.

Examples of pharmaceutically and veterinarily acceptable carriers ordiluents are demineralised or distilled water; saline solution;vegetable based oils such as peanut oil, safflower oil, olive oil,cottonseed oil, maize oil, sesame oils such as peanut oil, saffloweroil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil orcoconut oil; silicone oils, including polysiloxanes, such as methylpolysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;volatile silicones; mineral oils such as liquid paraffin, soft paraffinor squalane; cellulose derivatives such as methyl cellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol; lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3butylene glycol or glycerin: fattyacid esters such as isopropyl palmitate, isopropyl myristate or ethyloleate; polyvinylpyrridone; agar; carrageenan, gum tragacanth or gumacacia, and petroleum jelly, Typically, the carrier or canters will formfrom 10% to 99.9% by weight of the compositions.

In a preferred form the pharmaceutical composition of a compoundsuitable for use in the invention comprises an effective amount of anactive agent, together with a pharmaceutically acceptable canter,diluent and/or adjuvant as shown in Example 6.

The pharmaceutical compositions representing a component of the systemof the invention may be administered by standard routes. in general, thecompositions may be administered by the topical, transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, oral, rectal or parenteral (e.g.,intravenous, intraspinal, subcutaneous or intramuscular route. Stillgenerally, the compositions representing a component of the system ofthe invention may be in the form of a capsule suitable for oralingestion, in the form of an ointment, cream or lotion suitable fortopical administration, in a form suitable for delivery as an eye drop,in an aerosol form suitable for administration by inhalation, such as byintranasal inhalation or oral inhalation.

For administration as an injectable solution or suspension, non-toxicparentally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and1,2-propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvantsfor oral use include peanut oil, liquid paraffin, sodiumcarboxymethylcellulose, methylcellulose, sodium alginate, gum acacia,gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine andlecithin. In addition these oral formulations may contain suitableflavouring and colourings agents. When used in capsule form the capsulesmay be coated with compounds such as glycel, monostearate or glyceryldistearate which delay disintegration of the capsule.,

Adjuvants typically include emollients, emulsiflers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinyopyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols zein, shellac or gluten, Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulfite. Suitable lubricants includemagnesium stearate, saric acid, sodium oleate, sodium chloride or talc.Suitable time delay agents include glyceryl monostearate or glyceryldistearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides, or. mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, poly-vinyl-pyrrolidone, sodium alginate oracetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene to sorbitan mono- or di-oleate, -stearate or -laurate,and the like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above, or natural gums such as guar gum, gumacacia or gum tragacanth.

The topical formulations for use in the present invention, comprise anactive ingredient together with one or more acceptable carriers, andoptionally any other therapeutic ingredients.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops for use in the present invention may comprise sterile aqueous oroily solutions or suspensions. These may be prepared by dissolving theactive ingredient in an aqueous solution of a bactericidal and/orfungicidal agent and/or any other suitable preservative, and optionallyincluding a surface active agent. The resulting solution may then beclarified by filtration, transferred to a suitable container andsterilised. Sterilisaton may be achieved by: autoclaving or maintainingat 90° C.-100° C. for half an hour, or by filtration, followed bytransfer to a container by an aseptic technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the dropsare phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorheidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those described above in relation to thepreparation of drops. Lotions or liniments for application to the skinmay also include an agent to hasten drying and to cool the skin, such asan alcohol or acetone, and/or a moisturiser such as glycerol, or oilsuch as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention aresemisolid formulations of the active ingredient for externalapplication, They may be prepared by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with a greasy or non-greasy basis. Thebasis may comprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap; a mucilage; an oil of natural originsuch as almond,. corn, arachis, castor or olive oil; wool fat or itsderivatives, or a fatty acid such as stearic or oleic acid together withan alcohol such as propylene glycol or macrogols.

The formulation may incorporate any suitable surface active agent, suchas an anionic, cationic or non-ionic surface active such as sorbitanesters or polyoxyethylene derivatives thereof. Suspending agents such asnatural gums, cellulose derivatives or inorganic materials such assilicaceous silicas, and other ingredients such as lanolin, may also beincluded.

The compositions for parenteral administration will commonly comprise asolution of an active agent useful a component of the system of thepresent invention or a cocktail thereof dissolved in an acceptablecarrier, such as water, buffered water, 0.4% saline, and 0.3% glycineetc, wherein such solutions are sterile and relatively free ofparticulate matter.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The pharmaceutical compositions representing a component and/or activeagent of the system of the invention may also be administered in theform of liposomes. Uposomes are generally derived from phospholipids orother lipid substances, and are formed by mono- or multi-lamellarhydrated liquid crystals that are dispersed in an aqueous medium. Anynon-toxic, physiologically acceptable and metabolisable lipid capable offorming liposomes can be used. The formulations in liposome form maycontain stabillsers, preservatives, excipients and the like. Thepreferred lipids are the phospholipids and the phosphatidylcholines(lecithin), both natural and synthetic. Methods to form liposomes areknown in the art, and in relation to this specific reference is made to:Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq., the contents of which is incorporatedherein by reference.

4. Kits

Systems suitable for use in accordance with the present invention can beavailable as kits whereby respective components of the system arepackaged separately, Suitable containers Include glass containers,plastic containers, and strips of plastic or paper such as blisterpacks, The kits typically include containers for housing the variuscomponents and instructions for using the kit components in accordancewith the methods of the present invention.

The kits can be used in accordance with the methods of treatment ormethods of detection of the present invention.

An example of one kit according to th present invention is used forinducing thrombosis of tumour vasculature and necrosis of a tumour. Thekit includes a first container containing GSAO-B and a second containercontaining avidin and a third container containing tTF-B. The GSAO-B isfirst administered to a vertebrate, followed by an interim period,typically from 12 to 24 hours, still typically about 18 hours. Thesecond component, avidin, is combined with the third component, tTF-Band the resultant avidin-tTF-B complex administered to the vertebrate.This kit allows GSAO-B to target apoptotic cells such that when theavidin-tTF-B complex is administered, the tT-B binds to the GSAO-B atapoptotic cells and induces thrombosis.

5. Treatment and/for Prevention of Disease

The systems for use in the invention are useful in the treatment ofvarious disorders and diseases of vertebrates. Examples of disorders anddiseases may be grouped into broad categories such as the following:angiogenesis-dependent diseases, cellular proliferative diseases (e.g.psoriasis, IBD, malignancies, restenosis), inflammatory disorders,auto-immune diseases, blood vessel diseases, thrombosis, cancer,neurodegenerative disorders (e.g Alzhelmer's disease, Parkinson'sdisease), myelodysplastic syndromes, ischaemia/repurfusion injury andorgan transplant injury.

Typically, the cancer is selected from the group consisting ofcarcinogenic tumours, tumours of epithelial origin, such as cola-rectalcancer, breast cancer, lung cancer, head and neck tumours, hepaticcancer, pancreatic cancer, ovarian cancer, gastric cancer, brain cancer,bladder cancer, prostate cancer and urinary/genital tract cancer;mesenchymal tumours, such as sarcoma; and haemopoletic tumours such as Bcell lymphoma.

Typically, the cancer is a haematological tumour. More typically, thecancer is a solid tumour,

The systems of the invention may also be used in the treatment ofinflammatory disorders and/or auto-immune diseases, examples of whichinclude the following: rheumatoid arthritis, seronegative arthritidesand other inflammatory arthritides, systemic lupus erythematosus,polyarteritis and related syndromes, systemic sclerosis, Sjögren'ssyndrome and other inflammatory eye disease, mixed connective tissuedisease, polymyositis and dermatomyositis, polymyalgia rheumatica andgiant cell arteritis, inflammatory joint disease, non-inflammatoryarthropathies and soft tissue rheumatism, algodystrophy.

Examples of blood vessel disease and thrombosis for which the systems ofthe invention are useful in a preventive manner and/or in the treatmentof, include the following: progression of atherosclerosis;cerebrovascular accidents such as transient ischaemic, completed stroke,and after carotid surgery; acute myocardial infarction (primary andsecondary); angina; occlusion of coronary artery bypass graft occlusionfollowing percutaneous transluminal coronary angioplasty: occlusionfollowing coronary stenting; vascular occlusion in peripheral arterialdisease; venous thromboembolic disease following surgery, or duringpregnancy, or during immobilisation.

Examples of small vessel disease for which the systems of the inventionare useful include the following: glomerulonephritis; thromboticthrombocytopenic purpura; the haemolytic uraemic syndrome; placentalinsufficiency and preeclampsia.

The systems of the invention may also be used for the treatment ofvascular syndromes and myeloproliferative diseases.

The systems of the invention may also find use in the prevention ofthrombosis formation in the following situations: artificial/prostheticvascular shunts and grafts; prosthetic heart valves; cardiopulmonarybypass procedures; haemoperfision and haemodialysis.

Typically, the systems of the invention may be used in combination withother known treatments, such as surgery and/or therapeutic agents,including chemotherapeutic or radiotherapeutics. For example, when usedin the treatment of solid tumours, compounds of the present inventionmay be administered with chemotherapeutic agents such as: adriamycin,taxol, fuorouricil, melphalan, cisplatin, alpha interferon, COMP(cyclophosphamide, vincristine, methotrexate and prednisone), etoposide,mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide,vincristine and dexamethasone), PROMACE/MOPP (prednisone, methotrexate(w/feucovin rescue), doxorubicin, cyclophosphamide, taxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP470, pentosan polysulfate,platelet factor 4, angiostatin, LM609, SU-101. CM-101, Techgalan,thalidomide, SP-PG and the like. Other chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphan, chlorambucil, cyclophosphamide and ifosfamide; nitrosoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates including busulfan; triaznes including dacarbazine;ethyenimines including thlotepa and hexamethyimelamine; folic acidanalogues including methotrexate; pyrimidine analogues including5-fuorouracil, cytosine arabinoside; purine analogues including 6mercaptopurine and 6-thioguanine; antitumour antibiotics includingactinomycin D; the anthracyclines including doxorubicin, bleomycin,mitomycin C and methramycin; hormones and hormone antagonists includingtamoxifen and cortiosteroids and miscellaneous agents includingcisplatin and brequinar.

Typically, the physiological system to be treated in accordance with thepresent invention (e.g, the hepatic system, pancreatic system) may beisolated by or during surgery prior to administration of the system ofthe invention.

The invention will now be described in greater detail by reference tospecific Examples which should not be construed as in any way limitingthe scope of the invention.

EXAMPLE 1 Synthesis of Arsenoxide Compounds

The following chemicals were purchased and used without furtherpurification. phenylarsenoxide, bromoacetyl bromide, sulfur dioxide,d₆-dimethylsulfoxide, deuterium oxide, methanol, 98% sulfuric acid, 48%hydrobromic acid, 37% hydrochloric acid (Ajax; Auburn, NSW);dichloromethane, potassium hydroxide, sodium hydrogen carbonate, sodiumhydroxide (BDH, Kilsyth, VIC); P-2 Gel extra fine 1,800 MW cut-off(Bio-Rad, Hercules, Calif.); 2,3dimercaptopropanol (DMP); thionylchloride (Merck, Darmstadt, Germany); 6,8-thioctic acid,ethylenediaminetetraacetic acid, N(2-hydroxyethyl)piperazine-N′2ethanesulfonic acid), sodium carbonate, sodium chloride, sodium iodide(Sigma, Castle Hill, NSW); p-arsanilic acid (Tokyo Kasei Kogyo, Tokyo,Japan); glycine (ICN, Aurora, Ohio);6-((6-((biotinoyl)amino)hexanoyl)amino)hexanoic acid, succinimidyl ester(biotin-X)(, SE) were obtained from Molecular Probes, Eugene, Oreg., Allother reagents were of analytical grade.

Instrumentation—1D and 2D NMR spectra were obtained using a BrukerDPX300 nuclear magnetic resonance spectrometer, with ¹H-and ¹³C detectedat 300.17 MHz and 75.48 MHz, respectively. UV-visible absorbances wererecorded on a Molecular Devices Thermomax Plus (Palo Alto, Calif.)microplate reader.

Preparation of acidified deuterium oxide—Fresh thionyl chloride wascautiously added to an excess of deuterium oxide. After evolution of SO₂had ceased, the resulting solution (0.6 ml) was added to GSAO (ca 50 mg)in a 5 mm NMR tube. This sample was used to obtain the NMR spectra.

Example 1(a) Synthesis of4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO)

The total synthesis of GSAO is represented schematically in FIG. 1.

Synthesis of 4-(N-bromoacetyl)amino)phenylaronic acid (BRAA)

Sodium carbonate (40.14 g, 378.7 mmol) was added to water (200 mL) andstirs at room temperature until all solids had dissolved, To the stirredcarbonate solution was added p-arsanillc acid (29.99 g, 138.2 mmol),portionwise, and the volume of the solution made up to 300 mL withaddition of more water. The solution (pH 10 to 11) was allowed to stirfor 30 mins, and if necessary, was filtered to remove any undissolvedsolid before being refrigerated for 2 to 3 hours. The solution wastransferred to a separating funnel and ice chips were added. Bromoacetylbromide (15 mL, 34.76 g, 172.1 mmol) was diluted in dichloromethane (50mL) and approximately half of the dichloromethane solution was addedcarefully to the cold aqueous solution. The mixture was cautiouslyshaken, with frequent venting to avoid excessive build up of pressure.After 1 to 2 mins, the evolution of carbon dioxide had subsided, andmore vigorous shaking was undertaken, The remaining portion ofbromoacetyl bromide was carefully added and the procedure repeated. Whenthe reaction was over, the solution was found to be pH 7. The lowerdichloromethane layer was discarded, and the aqueous layer transferredto a 1 L flask and carefully acidified by dropwise addition of 98%sulfuric acid. Complete precipitation of the white product requiredaddition of acid until the solution was approximately pH 1. The crudeproduct was collected and dried at the pump, typically in yields of 50%to 75%. ¹H-NMR (d₆DMSO): δ4.09 (s, 2H), 7.73 (d, J=9 Hz, 2H), 7.83 (d,J=9 Hz, 2H), 10.87 (s, 1H). ¹³C-NMR (d₆-DMSO); δ30.53, 119.97, 127.34,131.56, 143.08, 166.00 ppm.

Synthesis of 4(N-bromoacetyl)amino)phenylarsenoxide hydrate (BRAO.xH₂O)

Into a 3necked 500 mL round-bottomed flask was placed BRAA (12.15 g, 36mmol). The solid was dissolved with swirling in a mixture of methanol(75 mL) and hydrobromic acid (48%, 75 mL), giving a transparent yellowsolution. The solution was filtered to remove residual solids. SodiumIodide (0.20 g, 1.3 mmol was added as a catalyst, whereupon the colourof the solution darkened to orange-brown, then sulfur dioxide gas wasslowly (ca. 2 bubbles per second) passed through the stirred solutionfor approximately 2.5 hours. The resultant white precipitate wascollected using a Büchner funnel, giving the product (17.43 g) as a dampwhite solid. The activity of a solution made by dissolving a portion ofthe solid (40.7 mg) in deoxygenated DMSO (800 μL) was determined to be56 mM (see below). Hence, the molecular weight of BRAO.xH₂is 908.5, thatis, 35% w/w BRAO and 65% w/w H₂O. Therefore, the “anhydrous” weight ofthe BRAO product was 35% of 17.43 g, that is, 6.10 g (19 mmol, 53%yield). ¹H-NMR (d6DMSO):δ4.85 (s, 2H), 7.78 (d, J=9 Hz, 2H) 7.86 (d, J=9Hz, 2H), 11.36 (s, 1H). ¹³C-NMR (d₆-DMSO): δ30.55, 119.22, 130.52,140.04, 145.04, 165.52 ppm.

Synthesis of 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO)

DMSO (10 mL) was deoxygenated by passing a stream of nitrogen gasthrough it for a few minutes, and used to dissolve BRAO.xH₂O (1.00 g,248 mmol active arsenoxide). Glutathione (1.15 g, 3.74 mmol, 1.5 eq) wasdissolved in 0.5 M bicarbonate buffer, pH 9.6 (35 mL), and added to thesolution of BRAO.xH₂O in DMSO. The total volume was made up to 50 mLwith 0.5 M bicarbonate buffer, and the solution gently agitated at roomtemperature overnight. Cautious neutralisaton with 37% hydrochloricacid, followed by lyophilisation gave a white powdery product, whichcould be dissolved in water leaving no residual solid. The activearsenoxide concentration of the resultant solution was found to be 49.6mM, determined using the DMP/DTNB assay (see below).

The product was purified using gel-filtration (P-2 Gel extra fine, 1.8kDa cutoff, 50 g) on a 130 mL column, using 20 mM Hepes, 0.14 M NaCl, 1mM EDTA, pH 7.4 buffer as the eluant at a flow rate of 0.10 mL/min. Atotal of 144 mL was collected (72 fractions of 2 mL) and monitored by UV(λ214 nm). Four peaks, A, B, C and D, were resolved. Peaks B and Cshowed activity in the DTNB/DMP assay (see below), and were assigned asGSAO and unreacted BRAO, respecively. Peaks A and D were tentativelyassigned as the oxidation products GSSM and BRAA (the oxidation productof BRAO), respectively (see below). Unreacted glutathione was alsodetected (using DTNB) in the fractions corresponding to Peak A. Thefractions corresponding to peak B were combined and deoxygenated withnitrogen gas to give a solution of GSAO (15 mM, approximately 12 mL).¹H-NMR (D₂O): δ1.93 (q, J=7 Hz, 2H), 2.35 (t,J 8 Hz, 2H), 2.84 (dd, J=14Hz, J=9 Hz, 1H), 3.05 (dd, J=14 Hz, J =5 Hz, 1H), 3.35 (s, 2H), 3.58(t,J 6 Hz, 1H), 3.64 (d,J=2 Hz, 2H), 4.48 (dd, J =9 Hz, J =5 Hz, 1H),7.44 (d,J=8 Hz, 2H), 7.58 (d,J=8 Hz, 2H). ¹³C-NMR (D₂O): δ25.93, 31.16,33.53, 36.01, 42.97, 52.83, 53.89, 121.29, 129,97, 138.77, 144.09,170.90, 171.73, 173.75, 174.68, 175.76 ppm.

2D NMR spectroscopy was also used to confirm the structure of GSAO. Aseries of 1H and 13C NMR spectra, ¹H, 13C, ¹H—¹H COSY, 1H—¹³C HMQC and¹H—¹³C HMBC, were all found to be consistent with the structure proposedin FIG. 1. Considered together, all of the spectra permitted theunambiguous assignment of all carbon and non-exchangeable hydrogenatoms. An expansion of the 1H—¹³C HMBC spectrum of GSAO, showing thealiphatic region, is shown in FIG. 2. The ¹H—¹³C HMBC techniquecorrelates coupled ¹H and ¹³C nuclei, but filters out directly bondednuclei. This means that ¹H and ¹³C nuclei that are separated by two,three, or (sometimes) four bonds appeared as crosspeaks in the spectrum.FIG. 2 shows that C11 is only strongly coupled to H7 (referring to theprotons attached to C7), while C7 is strongly coupled to H11 in additionto H6. This confirms that the glutathione sulfur was successfullyalkylated with BRAO.

Example 1(b) Synthesis of 4-(N-(S-qlutathionylacetyl)amino)phenylarsonicacid (GSAA)

The synthesis of GSAA is represented schematically in FIG. 3.

BRAA (1.00 g, 296 mmol) and glutathione (1.36 g, 4.44 mmol, 1.5 eq) weredissolved in 0.5 M bicarbonate buffer, pH 9.6 (50 mL), and the solutiongently agitated at room temperature overnight. Lyophilisation gave awhite powdery product which was freely soluble in water, leaving nosolid residue. The product was purified by gel-filtration on a 570 mLcolumn (2.5×117 cm) of Bio-Gel P-2 extra fine (BioRad, Hercules, Calif.)using deionised water as the eluant at a flow rate of 0. 1 mL per min.The product (GSAA) eluted from the column in a position corresponding toPeak A in the purification of GSAO.

Example 1(c)

Synthesis of4-(N-(S-(N-(6-((6-biotinoyl)amino)hexanoyl)amino)hexanoyl)glutathionyl)-acetyl)-amino)phenylarsenoxide(GSAO-B)

The synthesis of GSAO-B is represented schematically in FIG. 4, GSAO(0.13 g) was dissolved in 0.5 M sodium bicarbonate buffer (5 mL, pH 8.5)and the concentration of active arsenical in the resultant solution wasdetermined to be 39 mM. The buffered arsenical solution (4.2 mL,containing 165 μmol active arsenical) was added to a solution ofbiotin-XX, SE (100 mg. 176 μmol in DMSO (1 mL), the mixture inverted afew times and then incubated at 4° C. for 4 hours. Glycine (17.5 mg, 233μmol) was added and the mixture kept at 4° C. overnight. Theconcentration of trivalent arsenical in the GSAO-B product wasdetermined to be 31 mM and the solution was used without furthermodification.

Example 1(d) Conjugation of Fluorescein to GSAO or GSAA

A solution of fluorescein-5-EX succinimidyl ester (Molecular Probes,Eugene, Oreg.) (2.4 mg, 4.1 μmol) in DMSO (240 μL) was added to GSAO orGSAA (33.8 mM) in Mes buffer, pH 5.5 (5 mM, 473 μL), and the mixture wasdiluted with bicarbonate buffer, pH 9 (0.5 M, 3.287 mL) and allowed tostand at room temperature for 80 min. The reaction was then diluted withglycine (100 mM) in PBS (4 mL), and allowed to stand at room temperatureovernight The final solution contained trivalent arsenical (2.00 mM) andglycine (50 mM. The molar ratio of fluorescein-5× to GSAO or GSAA was˜1.5:1. The molecular weights of GSAO- and GSAA-fluorescein (GSAO-F andGSAA-F) are 1024 and 1040, respectively. The synthesis of GSAO-F isrepresented schematically in FIG. 5.

Example (1e) Conjugation of Cy™5.5 to GSAO or GSAA

A solution of Cy™5.5 (Amersham Pharmacia Biotech, Uppsala, Sweden) (266nmol) in bicarbonate buffer, pH 9 (0.5 M, 968 μL) was mixed with a solonof GSAO or GSAA (33.8 mM) in Mes buffer, pH 5.5 (5 mM. 32 μL), andallowed to stand at room temperature for 80 min. The reaction was thendiluted with glycine (100 mM) in PBS (1 mL), and allowed to stand atroom temperature overnight The final solution contained trivalentarsenical (0.54 mM) and glycine (50 mM). The molar ratio of GSAO orGSAA-Cy™5.5 was ˜4:1. The molecular 9 e o GSAO-Cy™5.5 and GSAA-Cy™5.5are 1447 and 1463, respectively. The synthesis of GSAO-Cy ™5.5 isrepresented schematically in FIG. 6.

Example 2 Example 2(a) Assay of GSAO-B, GSAO-F and GSAO-Cy™5.5

Concentrations of GSAO-B, GSAO-fluorescein (GSAO-F) and GSAO-Cy™5.5 insolution were measured by filtrating with dimercaptopropanol (DMP) andcalculating the remaining free thiols with 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB) (Sigma, St. Louis, Mo.) (Donoghue et al., 2000). A stocksolution of DMP (5 μL, 50 μmol was dissolved in DMSO (995 μL), giving aconcentration of 50 mM DMP. A second dilution of the 50 mM DMP stocksolution (10 μL) in pH 7.0 buffer (0.1 M HEPES, 0.3 M NaCl, 1, mM EDTA)(990 μL) gave a working solution of 500 μM DMP. The activity of thearsenical could then be determined by the titration of varying amountsof arsenical against the DMP working solution (10 μL) in a 96-wellmicrotitre plate, with the total volume made up to 195 μL by addition ofbuffer. After a 10 minute incubation at room temperature, during whichtime the solutions were agitated on a plate shaker, 5 μL of a 37.9 mMthe solution of DTNB (15 mg) in DMSO (1 mL) was added, and the plateincubated with shaking for another 10 minutes. The absorbance at 412 nmdue to the formation of the TNB dianion was measured using a MolecularDevices Thermomax Plus (Palo Alto, Calif.) microplate reader. Theextinction coefficient for the TNB dianion at pH 7.0 is 14,150 M⁻¹cm⁻¹at 412 nm (Riddles et al., 1983). The conjugates were sterile filteredand stored at 4° C. in the dark until use. There was no significant lossin the active concentration of stock solutions of the arsenicals for atleast a week when stored under these conditions. The glycine slows theoxidation of GSAO to GSAA (Donoghue et al, 2000).

Example 2(b) Interaction of GSAO-B with PDI and thloredoxin

Human recombinant PDI and thloredoxin bound GSAO-B (FIG. 7). Recombinanthuman protein disulfide isomerase (PDI) was produced in E. coil andpurified according to Jiang et al. (1999). In the experiment, purifiedPDI, thloredoxin or albumin as negative control were incubated with a2-fold molar excess of dithiothreitol for 60 minutes to ensure that theactive site disulfides of PDI and thloredoxin were in the reduceddithiol state. The proteins were then incubated with GSAO-B or GSAO-Band a 4-fold molar excess of DMP for 30 minutes. Equivalent moles of thelabelled proteins were resolved on SDS-PAGE, transferred to PVDFmembrane, and blotted with streptavidin-peroxidase to detect the GSAO-Blabel. Samples were resolved on 4-15% SD$PAGE under non-reducingconditions and transferred to PVDF membrane. Proteins were detected byWestern blot using an anti-PDI murine monoclonal antibody (Jiang et al.,1999) (used at 2 μg per mL). Rabbit anti-mouse horseradish peroxidaseconjugated antibodies (Dako Corporalon, Carpinteria, Calif.) were usedat 1:2000 dilution. GSAO-B-labelled proteins were blotted withstreptavidin peroxidase (Amersham, Sydney, NSW) used at 1:1000 dilution.Proteins were visualised using chemiluminescence (DuPont NEN, Boston,Mass.) according to the manufacture's instructions. Chemiluminescencefilms were analysed using a GS-700 Imaging Densitometer andMulti-Analyst software.(Bio-Rad, Hercules, Calif.).

Both PDI and thioredoxin incorporated GSAO-B but albumin did not Thehigher M_(r) band in lane 1 of FIG. 7B was a small amount of aggregatedPDI in the preparation (Jiang et al., 1999). It is noteworthy that thedensity of labelling of PDI was approximately twice that of thioredoxinwhich is consistent with the two active site dithiols of PDI versus theone of thioredoxin.

EXAMPLE 3 In vitro I In vivo Studies Example 3(a) Staining of AdherentCells with GSAO-B

Methods

BAE cells were seeded at a density of 5×10⁵ cells per well in 2-wellglass chamber slides (Nunc, Naperville, Fla.) and allowed to attachovernight Cells were treated for 16 h with 10 μM GSAO-B, then washedtwice with PBS, bed in PBS containing 3.7 % formaldehyde, permeabilisedwith PBS containing 3.7 % formaldehyde and 0.1 % TritonX-100, thenwashed twice and incubated for 1 h at room temperature with a 1:200dilution of Alexa 488conjugated streptavidin (Molecular Probes, Eugene,Oreg.) in PBS containing 1% BSA. Cells were then washed three times withPBS and mounted in VectaShield antifade agent (Vector Laboratories,Burlingame, Calif.). Pictures were taken with an Olympus BX60 microscopewith BX-FLA fluorescence and a Diagnostic Instruments Spot DigitalCamera and software v2.2 (Stewing Heights, Mich.).

Results

When cultured endothelial cells were stained with GSAO-B and visualisedwith streptavidin-Alexa 488 it was observed that the occasional cellstained very brightly (FIG. 8). The vast majority of the cells boundvery little GSAO-B.

Example 3(b) GSAO Labelled Apoptotic Cells Following Caspase Activation

Methods

Cell Culture

HT1080 human fibrosarcoma and bovine aortic endothelial cells (ATCC,Bethesda, Md.) were cultured in DMEM containing 10% FBS, 2 mML-glutamine, and 5 U.mL⁻¹ penicillin/streptomycin (Gibco BRL,Gaithersburg, Md.). The human microvascular endothelial cell line(HMEC-1) (Ades et al., 1992) was cultured in MCDB131 medium (Gibco BRL,Gaithersburg, Md.) containing 10 % foetal bovine serum, 2 mML-glutamine, 5 U.mL⁻¹ penicillin/streptomycin, 10 ngml⁻¹ epidermalgrowth factor (Gibco BRL, Gaithersburg, Md.) and 1 μgml¹ hydrocortisone(Sigma, St. Louis, Mo.). Cells were detached with PBS containing 10 mMEDTA or with a trypsin/EDTA solution (Gibco BRL, Gaithersburg, Md.).Culture plates were from Coming Costar, Coming, N.Y.

Flow Cytometry of GSAO- and Annexin V-labeled Cells

HT1080 cells were treated for 20 h with 1 μg.mL⁻¹ camptothecin(Calbiochem, San Diego, Calif.), then detached with PBS containing 2.5mM EDTA and combined with cells that had detached during incubation. Thecells were washed twice with annexin V binding buffer (10 mM Hepes, 140mM NaCl, 2.5 mM CaCl₂, pH 7.4), and 2×10⁵ cells per treatment wereincubated in 100 μL annexin V binding buffer containing 10 μM GSAO-F orGSAA-F for 15 min at room temperature with shaking. The cells werewashed twice, then incubated for 15 min in 100 μL annexin V bindingbuffer containing 5 μl phycoerythrin-conjugated annexin V (annexin V-PE)(PharMingen, San Diego, Calif.) and 1 μl of a 100 μg.mL⁻¹ solution ofpropidium iodide (Molecular Probes, Eugene, Oreg.). The total volume wasthen made up to 500 μl with annexin V binding buffer, and the sampleswere transferred to ice. Flow cytometry was performed using a FACSstarflow cytometer (Beckton Dickson). Results are derived from 10⁴ cells persample. To test the effect of caspase inhibition on to GSAO-F uptake,cells were treated with camptothecin in the absence or presence of 10μg.mL⁻¹ Z-VAD-FMK(carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone)(Calbiochem-Novabiochem, San Diego, Calif.), then detached, washed andincubated in annexin V binding buffer containing 10 μM GSAO-F for 15min. The cells were washed twice and subjected to flow cytometry.

Results

Human fibrosarcoma HT1080 cells were treated with the topolsomeraseinhibitor, camptothecin (Kaufmann, 1998), for 20 h to induce apoptosis,and untreated or camptothecin-treated cells were incubated with acombination of fluorescein-conjugatd GSAO (GSAO-F) or GSAA (GSAA-F) andphycoerythrin-conjugated annexin V (annexin V-PE). The fluorescence wasquantitated by flow cytometry (FIG. 9 a-f) An increase in apoptotic anddead cells after camptothecin treatment was apparent, with a 7.3-foldincrease in the proportion of cells that had a phycoerythrinfluorescence of greater than 100 fluorescence units. This increase inannexin V-positive cells correlated with an 8.6-fold increase in theproportion of cells whose fluorescein fluorescence was greater than 1027units (FIG. 9 c). By contrast, camptothecin treatment did not increasethe proportion of cells that co-labelled with GSAA-F and annexin V-PE(FIG. 9 f. The mean fluorescein fluorescence of untreated cells labelledwith GSAA-F or GSAO-F was similar (255 units for GSAO-F; 213 units forGSAA-F), whilst for the camptothecin-treated cell population a cleardifference in mean fluorescence was apparent (1400 units for GSAO-F, 219units for GSAA-F). Similar results were obtained when apoptosis wasinduced in human dermal microvascular endothelial calls using either 1μg.mL⁻¹ camptothecin or 10 or 25 mM homocysteine for 24 h (not shown).

HT1080 cells were left untreated or treated with camptothecin to induceapoptosis, then detached and labelled with GSAO-F, annexin V-PE and thenucleic acid binding dye, propidium iodide. Propidium iodide is taken-upby cells in the later stages of apoptosis and by necrotic cells, whilstannexin V binds to cells early in the apoptotic program. Annexin V-PEfluorescence was plotted against GSAO-F fluorescence for all cells, andall cells excluding those that had taken-up propidium iodide (>200fluorescence units). The percentage of cells that stained brightly withboth annexin V-PE (>200 fluorescence units) and GSAO-F (>1980fluorescence units) are shown (FIG. 9 g). GSAO bound to both apoptoticand dead cells.

The caspases are a class of aspartate proteases that are activated byapoptotic stimuli in all cells, triggering the proteolysis of cellulartargets such as the cytoskeleton and the fragmentation of nuclear DNA(Thornberry and Lazebnik, 1998). The activation of caspases is anessential component of the apoptotic program, and the broad-spectrumcaspase inhibitor Z-VAD-FMK has been used extensively to blockcaspase-dependent processes (Zhu et al., 1995). HT1080 cells weretreated with camptothecin as above, in the presence or absence ofZ-VAD-FMK Treatment with Z-VAD-FMK substantially blocked the increase inGSAO-F positive cells seen with camptothecin treatment, indicating thatcaspase activity is a requirement for the uptake of GSAO Into apoptoticcells.

Example 3(c) GSAO Entered Apoptotic Cells and Labelled ProteinsContaining Closely-spaced Dithiols

Methods

Confocal Microscopy of GSAO- and Annexin V-labelled Cells

HT1080 cells were treated for 20 h with 1 μg.mL⁻¹ camptothecin, thendetached with PBS containing 10 mM EDTA, washed with annexin V bindingbuffer, and incubated for 15 min in annexin V binding buffer containing10 μM GSAO-F and 1:20 (v/v) Alexa594conjugated annexin V (MolecularProbes, Eugene, Oreg.). The cells were then washed twice with annexin Vbinding buffer and pipetted onto a microscope slide. Transverse cellsections were captured using an Olympus BX60 microscope and an OptiscanF900e confocal unit and software (Optiscan, Notting Hill, Austrlia).

Labelling of Cells with GSAO-B

HT1080 cells were for 20 h with 1 μg.mL⁻¹ camptothecin and labeled with10 μM GSAO and annexin V-PE as described above for labelling with GSAO-Fand annexin V-PE. Cells were sorted on the basis of their annexin V-PEfluorescence, washed three times with PBS and 4×10⁴ cells were lysed in40 μL of RIPA buffer (50 mM Tris-HCl, 0.5 M NaCl, 1% Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 10 μM leupeptin, 10 μM aprotinin, 2 mMphenylmethylsulfonyl fluoride, 5 mM EDTA, pH 8.0) at 4° C. Equal amountsof lysate were electrophoresed on an 8-16% gradient SDS-polyacrylamidegel (Gradipore, Sydney), transferred to polyvinyldiethylene fluoride(PVDF) membrane (Millipore, Bedford, Mass.) and biotinylated proteinswere detected with a 1.2000 dilution of avidin-peroxidase (MolecularProbes, Eugene, Oreg.). To identify proteins that had incorporatedGSAO-B, 20 μl of RIPA lysate was incubated with 5 μlstreptavidin-dynabeads (Dynal Biotech, Oslo, Norway) for 15 min at 40°C. The beads were then washed twice with RIPA buffer, resuspended in 20μl SDS-PAGE loading buffer, resolved on SDS-PAGE, transferred topolyvinylidene difluoride membrane, and the PDI was detected by Westernblot using 2 μg.mL⁻¹ of anti-PDI polyclonal antibodies (Donoghue et al.,2000) and 1:2000 dilution of goat anti-rabbit peroxidase-conjugatedantibodies Dako Corporation, Carpinteria, Calif.). The blots weredeveloped using ECL enhanced chemiluminescence (NEN, Boston, Mass.).

Untreated and camptothecin-treated HT1080 cells were also labelled with10 μM GSAO-B in the absence or presence of 50 μM dimercaptopropanol for15 min at 20° C. with shaking. Cells were washed three times with PBSand 2×10⁵ cells lysed in 100 μL of RIPA buffer at 4° C. Lysates wereresolved on SDS-PAGE and blotted with avidin-peroxidase as describedabove.

Results

HT1080 cells were treated with camptothecin to induce apoptosis, thendetached and is labelled with GSAO-F and annexin V-Alexa-594. Cells wereimaged by confocal microscopy. GSAO-F distributed in the cytoplasm ofannexin V-positive cells (FIG. 10 a(i-vi)). There was negligible GSAO-Ffluorescence in cells that did not label with annexin V (FIG. 10 a(vii).

HT1080 cells were treated with camptothecin to induce apoptosis, thendetached and labelled with annexin V-PE and biotin-conjugated GSAO (GSAOB) (Donoghue et al., 2000). Cells were sorted into annexin V-positive(>100 fluorescence units) and annexin V-negative (<100 units)populations and equivalent numbers of cells from each population wereresolved on SDS-PAGE and blotted with streptavidin-peroxidase to detectthe GSAO-labelled proteins. Approximately seven proteins clearlyincorporated GSAO-B in annexin V-positive cells (FIG. 10 b). The levelof incorporation of GSAO-B into annexin V-negative cells was negligibleby comparison.

The GSAO-B-labelled proteins were also collected onstreptavidin-dynabeads, resolved on SDS-PAGE and Western blotted forprotein disulfide isomerase (PDI), a protein that is abundant in theendoplasmic reticulum and functions as a redox chaperone (Donoghue etal., 2000; Novia, 1999). The intensity of labelling of PDI in annexinV-positive cells was much higher than labelling of this protein inannexin V-negative cells (FIG. 10 c).

To confirm that GSAO-B was interacting with closely-spaced dithiols inapoptotic calls, HT1080 cells were untreated or treated withcamptothecin, then detached and labelled with GSAO-B In the absence orpresence of dimercaptopropanol (FIG. 10 d). This synthetic dithiolcompetes with protein dithiols for binding to GSAO-B (Donoghue et al.,2000). Again, there was much more labelling of the camptothecin-treatedpopulation than the untreated population, and the labelling of apoptoticcellular proteins by GSAO-B was ablated by incubating the cells with a5-fold molar excess of dimercaptopropanol.

Example 3(d) GSAO Labelled Apoptotic or Dead Tumour Cells Vivo

Methods

BALB/C-nude mice (Biological Resources Centre, University of New SouthWales, Sydney) bearing S.C. BxPC-3 tumours in the proximal dorsum weregiven 36 mg/kg GSAO-B or GSAA-B-in 0.2 mL of PBS containing 20 mMglycine by S.C. injection in the hind flank. Mice were sacrificed 6hours later and tumours were embedded in OCT compound (Sukura, Torrence,Calif.) and snap frozen in liquid nitrogen. 5 μM sections of the tumourswere fixed with acetone and stained with StreptABComplex/HRP (DakoCorporation, Carpinteria, Calif.) according to the manufacture'sinstructions and counterstained with haematoxylin. Sections were alsofixed with acetone/methanol, permeabilised with .0.1% Triton X-100, andstained with rabbit anti-activated caspase-3 antibody (Promega, Madison,Wis.)/goat anti-rabbit Texas Red and avidin-Alexa Fluor 488 (MolecularProbes, Eugene, Oreg.). Sections were counterstained with DAPI (Sigma,St. Louis, Mo.) and mounted with fluoromount-G (Southern Biotechnology,Birmingham, Ala.).

Results

Mice bearing S.C. BxPC3 tumours were given GSAO-B or GSAA-B by S.C.injecton at a site remote from the tumour. The mice were sacrificed 6hours later, the tumours excised and GSAO-B was detected withstreptavidin-peroxidase. There was an accumulation of GSAO-B in regionsof the tumour where apoptotic or dead cells were prevalent (FIG. 11 a).GSAO-B stained cells that showed visible signs of apoptosis, including acondensed nucleus and shrunken cytoplasm, but not healthy tumour cells.There was no staining of tumour tissue that had not been incubated withstreptavidin-peroxidase or of tumours from mice that had been givenGSAA-B (not shown). Labelling of apoptotic cells was confirmed bystaining sections for activated caspase-3 and GSAO-B. Activated caspase3 and GSAO-B co-localised, while GSAA-8 was not detected in the sections(FIG. 11 b). GSAO-B was also detected in apoptotic and necrotic regionsof AsPC-1 human pancreatic carcinoma tumours grown S.C. in SCID mice(not shown).

Example 4 In Vivo imaging of Tumours with a Near-infrared FluorescentDye-labelled GSAO

Methods

C57BL/6 (Jackson Labs, Bar Harbor, ME) mice bearing S.C. Lewis lungtumours, SCID (Massachusetts General Hospital, Boston, Mass.) bearingS.C. BxPC3 tumours or TRAMP (Greenberg et al., 1995) mice were given 0.8mg/kg GSAO-Cy™5.5, GSAA-Cy™5.5 or Cy™5.5 dye alone in 0.1 mL of PBScontaining 20 mM glycine by S.C. injection in the light hind flank. TheS.C. tumours were established in the proximal dorsum.

The imaging system utilised a Leica Microsystems fluorescence microscope(Allendale, N.J.) with a 100 W halogen lamp source and a filter systemfor CY™5.5 (Chroma Technology, Brattleboro, VT). Anaesthetised mice wererestrained in a right prof box and fluorescence detected by a 12-bitmonochrome charged coupled device (Photometrics, Tuscon, Ariz.).Exposure time was 8 seconds with images digitally acquired as 16-bitTiff files in IPLab (Scanalytics, Fairfax, Va.). White light images ofthe tumours were also acquired.

Results

Cy™5.5 Is a near-infrared fluorescent dye that has been used to imagetumours in vivo (Weissleder et al., 1999). C57BL/6 mice being −0.3 gmurine Lewis lung tumours were administered 0.8 mg/kg GSAO-Cy™5.5 byS.C. injection and fluorescence images were acquired starting at 1 hourand ending at 48 hours after injecton. The white light image of a Lewislung s tumour is shown in FIG. 11 c(A). The same view as seen throughthe near -infrared filter one hour after injection is shown in FIG. 11c(B). The images show tat the GSAO-Cy™5.5 had been absorbed into thevasculature and labelled the skin as well as the tumour. Fluorescenceimages of the dorsal sin and the tumour 24 hours after injection areshown in FIGS. 11 c(C) and 11 c(D), respectively. The GSAO-Cy™5.5 hadcleared from the majority of the vasculature and only the tumourremained labelled. Examination of the urine indicated excretion of theGSAO-Cy™5.5 less then one hour after injection, with a peak intensity at4-8 hours (not shown). Further examination of the tumours at 30, 36, 48hours demonstrated a peak tumour to background signal ro at 24 hourswith little signal remaining at 48 hours. Neither GSAA-Cy™5.5 nor Cy™5.5labelled Lewis lung tumours (not shown). Murine T241 fibrosarcomatumours grown on the dorsum of C57BL/6 mice were also imaged usingGSAO-Cy™5.5 (not shown).

Human tumours grown in immunodeficient mice were also labelled byGSAO-Cy™5.5 SCID mice bearing ˜0.3 g human BxPC-3 pancreatic carcinomatumours were administered 0.8 mg/kg GSAO-Cy™5.5 by S.C. injection andfluorescence images were acquired 1 and 24 hours after injecton. Anexcellent signal to background ratio for me tumour was again observed at24 hours using GSAO-Cy™5.5 (FIG. 11 d(C)). Neither GSAA-Cy™5.5 (FIG. 11d(B)) nor Cy™5.5 (FIG. 11 d(D)) labelled BxPC3 tumours. The liver, heartlungs, bladder and kidneys were removed and examined for GSAO-Cy™5.5Only the bladder and kidneys had any evidence of a near-infrared signal(not shown), in addition, human CRL-1973 embryonal tumours grown S.C. onthe dorsum of SCID mice and human LnCaP prostate cells grown in. (heprostate of SCID mice were also imaged using GSAO-Cy™5.5 (not shown).These results indicate that GSAO-Cy™5.5™ran image both murine and humantumours in different strains of mice.

GSAO-Cy™5.5 was also used to image spontaneous prostate tumours in TRAMPmice (Greenberg et al., 1995).. The animals were examined withultrasound to verify the presence of tumours prior to imaging. TRAMPmice bearing prostate tumours were administered 0.8 mg/kg GSAO-Cy™5.5 byS.C. injection in the upper flank. As the depth detection threshold ofthe currently used imaging system was ≦1 cm (Weissleder et al., 1999),the abdomen was opened to reveal the tumour. The light field is filledwith a lame tumour and a central vein (FIG. 11 e(A)). The tumour waslabelled with GSAO-Cy™5.5 while the central vein was riot labelled (FIG.11 e(B)).

Example5 Example 5(e) Labelling of tTF with MPB

Cloning and Mutation of tTF

Human TF cDNA was from Karen Fisher (Fisher et al., 1987). The primersused to amplify and mutate the extracellular domain of TF (tTF, residues1-219) were 5′-ATCAGGATCCGGCACTACAAATACTGTG-3′ (forward primer) and5′-ATCAGGATCCTTAACATCTGAATTCCCCTTTCTCCTG-3′ (reverse primer). Bothprimers contain a BamHI site outside the coding sequence to facilitatecloning. A TGT codon for cysteine is located at codon position 219 inthe reverse primer, which replaces the GM codon for glutamine. The Q219CtTF cDNA was amplified by PCR using DNA polymerase Pfx and cloned intothe pTrcHisA vector (Invitrogen, San Diego, Calif.) at the BamHI site.The integrity, of the tTF cDNA and the Q219C mutation was confirmed byautomatic sequencing, The vector construct was used to transform E. coliBL21 Star (Invitrogen, San Diego, Calif.).

Expression, purification and biotin labeling of tTF

Recombinant tTF was expressed as described by Jiang et al. (1999),purified by ProBond (Invitrogen, San Diego, Calif.) affinitychromatography and refolded as described by Stone et al. (1995). TheHis-tag was cleaved from the purified, refolded protein by incubation of1 unit of EKMax (Invitrogen, San Diego, Calif.) per 0.2 mg of tTF for 16hours at room temperature. The cleaved His-Tag was separated from thetTF by anion-exchange chromatography. The reaction was dialysed against20 mM Tris.HCl, pH 8.0 buffer and applied to a 1 mL Mono-Q column(Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with the samebuffer. The bound protein was resolved with a 0 to 0.5 M linear NaClgradient The tTF eluted at ˜0.4 M NaCl.

The number of thiols in tTF were measured using DTNB. The tTF (˜10 μM)was incubated with DTNB (˜1 mM) in 0.1 M HEPES, 0.3 M NaCl, 1 mM EDTA,pH 7.0 buffer for 10 min at room temperature and the TNB was measuredfrom the absorbance at 412 nm using a Molecular Devices Thermomax Plus(Palo Alto, Calif.) microplate reader. The extinction coefficient forthe TNB dianion at pH 7.0 is 14,150 M⁻¹cm⁻¹ at 412 nm (Riddles et al.,1983). The tTF was incubated with a 20fold molar excess of3-(N-maleimidylpropionyl)biocytin (MPB) (Molecular Probes Incorporated,Eugene, Oreg.) for 16 h at room temperature to label the engineered Cys.Unreacted MPB was removed by dialysis.

Purified tTF and MPB-labelled tTF (tTF-B) were electrophoresed on a8-16% gradient SDS-polyacrylamide gel (Gradipore, Sydney) and stainedwith Coomassie Brilliant Blue (Sigma, St. Louis, Mo.). On one occasionthe proteins were transferred to polyvinyldiethylene fluoride(Millipore, Bedford, Mass.) and blotted with a 1:2000 dilution ofavidin-peroxidase (Molecular Probes, Eugene, Oreg.) to detect the MPBlabel. The blot was developed using ECL enhanced chemiluminescence (NEN,Boston, Mass.).

The Q219C tTF is shown in FIG. 12A. The protein contained 0.77 mol ofthiol per mol of protein, which reduced b 0.13 mot of thiol per mol ofprotein upon reaction with MPB. Incorporation of MPB into Q219C tTF isshown in FIG. 12B.

Example 5(b) Formation of the GSAO-B-avidin-tTF-B Complex

ELISA for Measuring Formation of the GSAO-B-avidin-tTF-B Complex

Human recombinant protein disulfide isomerase (PDI) was produced asdescribed by Jiang et al. (1999). PDI (100 μl of 5 μg.mL⁻¹ in 0.1 MNaHCO₃, 0.02% azide, pH 8.3 buffer) was adsorbed to Nunc PolySorp 96well plates overnight at 4° C. In a humid environment Wells were washedonce With phosphate buffered saline (PBS) containing 0.05% Tween 20(PBS/Tween), nonspecific binding sites blocked by adding 200 μl of 3%BSA in PBS and incubating for 90 min at 37° C., and then washed twotimes with PBS/Tween. All the following incubations were for 30 min atroom temperature with orbital shaking and the wells were washed threetimes with PBS/Tween after each incubation. Dithiothreitol (100 μl of 10mM) was added to the wells to reduce the active site disulfides of PDI.GSAO-B (100 μl of 100 μM) was added to the wells to label the activesite dithiols of PDI. tTF-B (0.1 μM) and increasing concentrations ofavidin (Sigma, St. Louis, Mo.) (100 μl final volume) was added to thewells to form PDI-GSAO-B-avidin-tTF-B complexes. Murine anti-human TFmonoclonal antibody (American Diagnostica, Greenwich, Conn.) (100 μl of2 μg.mL⁻¹) was added to the wells to detect the bound tTF-B and boundmurine antibody was measured with rabbit anti-murineperoxidase-conjugated IgG (100 μl of 1:500 dilution). Peroxidaseactivity was measured with 100 μl of 0.003% H₂O₂, 1 mg.mL⁻¹2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 50 mM citrate,pH 4.5 buffer for 20 min at room temperature with orbital shaking.Absorbances were read at 405 nm using a Molecular Devices ThermomaxKinetic Microplate Reader (Molecular Devices Corporation, Calif., USA).Results were corrected for control wells not coated with PDI.

GSAO-B reacts with the active site dithiols of purified PDI and with PDIon the cell-surface (Donoghue et al., 2000). This interaction has beenused in a microtitre plate format to examine the formation ofGSAO-B-avidin-tTF-B complexes. PDI was immobilised in microtitre wells,labelled with GSAO-B and incubated with tTF-B and increasing molarratios of avidin. The bound tTF was detected using an anti-TF antibody.The optimal avidin:tTF-B molar ratio for formation ofGSAO-B-avidin-tTF-B complexes was ˜0.5:1 (FIG. 13).

Example 5(c) Thrombosis of Tumor Vasculature and Necrosis of the Tumourby S.C. Administration of GSAO-B Followed by I.V. Administration ofAvidin tTF-B

Female 6-8 week old Balb/c nude mice (Biological Resources Centre,University of New South Wales, Sydney) were injected S.C. with 2×10⁶AsPC-1 cells in 0.2 mL of PBS in the proximal dorsal midline. Tumourvolume was calculated using m relationship, a.b.².0.52, where a is thelongest and b the shortest diameter. Tumours and organs were fixed inBuffered Formalde-Fresh (Fisher Scientific, Fair Lawn, N.J.), embeddedin pain and 5 μm thick sections were cut and placed on glass slides.Sections were stained with haematoxylin and eosin,

SCID mice bearing ˜0.6 g As-PC1 tumours were given 7.5 mg/kg GSAO-B in0.15 mL of PBS containing 20 mM glycine by S.C. injecton in flank. TheGSAO-B was allowed to clear from the general circulation for 18 hoursand the mice were then given 1 mg/kg avidin-[tTF-B] (molar ratio 0.5:1)in 0.2 mL of saline by I.V. injection in the tail vein. The principle ofthis treatment regimen is shown in FIG. 14.

Approximately 75% of the tumour was visibly purple 2-4 hours afteradministration of avidin-tTF-B. The mice was sacrificed 10 days afteradministration of avidin-tTF-B and the tumour excised and examinedhistologically. There was marked thrombosis of the tumour vasculatureand necrosis of the treated tumour (FIG. 15). Only a peripheral layer oftumour cells remained viable. The weight of the treated mouse did notchange over the course of the experiment At the conclusion of theexperiment, the heart, lungs, liver, kidneys, and spleen of the treatedmouse were examined histologically. There was no signs of thrombosis inany of the organs (not shown).

Example 6

Pharmaceutical Formulations

The compounds used as the components of the systems of he presentinvention may be administered alone, although fit is preferable thatthey be administered as a pharmaceutical formulation. The activeingredient may comprise, for topical administration, from 0.001% to 10%by weight, and more typically from 1% to 5% by weight of theformulation, although it may comprise as much as 10% by weight

In accordance with the best mode of performing the invention providedherein, specific preferred pharmaceutical compositions used ascomponents of the systems of the present invention are outlined below.The following am to be construed as merely illustrative examples offormulations and not as a limitation of the scope of the presentinvention in any way.

Example 6(e)—Topical Cream Composition

A typical composition for delivery as a topical cream is below:GSAO-Cy ™ 5.5  1.0 g Polawax GP 200 25.0 g Lanolin Anhydrous  3.0 gWhite Beeswax  4.5 g Methyl hydroxybenzoate  0.1 g Deionised &sterilised Water to 100.0 g 

The polawax, beeswax and lanolin are heated together at 60° C., asolution of methyl hydroxybenzoate is added and homogenisation achievedusing high speed stirring. The temperature is then allowed to fall to60° C., The component of the present invention, in this example beingGSAO-Cy™5.5, is then added and dispersed throughout, and the compositionis allowed to cool with slow speed stirring.

Example 6(b)—Topical Lotion Composition

A typical composition for delivery as a topical lotion is outlinedbelow: GSAO-Cy ™ 5.5 1.2 g Sorbitan Monolaurate 0.8 g Polysorbate 200.7g Cetostearyl Alcohol 1.5 g Glycerin 7.0 g Methyl Hydroxybenzoate 0.4 gSterillsed Water about to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of th warat 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenised, allowed to cool with continuousstirring and the GSAO-Cy™5.5 is added as a suspension in the remainingwater. The whole suspension is stirred until homogenised.

Example 6(c)—Eye Drop Composition

A typical composition for delivery as an eye drop is outlined below:GSAO-Cy ™ 5.5 0.3 g Methyl Hydroxybenzoate 0.005 g PrapylHydroxybenzoate 0.06 g Purified Water about to 100.00 ml.

The methyl-and propyl hydroxybenzoates are dissolved in 70 ml purifiedwater at 75° C., and the resulting solution is allowed to cool.GSAO-Cy™5.5 is then added, and the solution sterilised by filtrationthrough a membrane filter (0.22 μm pore size), and aseptically packedinto sterile containers.

Example 6(d)—Composition for Inhalation Administration

For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mgof GSAO-Cy™5.5 with 0.5-8% by weight of a lubricating agent such aspolysorbate 85 or oleic acid, is dispersed in a propellant such asfreon, and put into an appropriate aerosol container for eitherIntranasal or oral inhalation administration.

Example 6(d)—Composition for Parenteral Administration

A pharmaceutical composition of the present invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, and1 mg of GSAO-Cy™5.5.

Similarly, a pharmaceutical composition for intravenous infusion maycomprise 250 ml of sterile Ringer's solution, and 5 mg of GSAO-Cy™5.5.

Example 6(f)—Capsule Composition

A pharmaceutical composition of GSAO-Cy™5.5 in the form of a capsule maybe prepared by filling a standard two-piece hard gelatin capsule with 50mg of GSAO-Cy™5.5, in powdered form, 100 mg of lace, 35 mg of talc and10 mg of magnesium stearate.

Example 6(g)—Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by Injecton may be prepared by mixing 1% by weight ofGSAO-Cy™5.5 in 10% by volume propylene glycol and water. The solution issterilised by filtration.

Example 6(h)—Ointment Composition

A typical composition for delivery as an ointment includes 1.0 g ofGSAO-Cy™5.5, together with white soft paraffin to 100.0 g, dispersed toproduce a smooth, homogeneous product.

1. A method of selectively targeting an active agent (or agent capableof becoming an active agent) to apoptotic cells, comprising contactingsaid cells with a system comprising an arsenoxide (or arsenoxideequivalent) compound and said agent, wherein said arsenoxide (orarsenoxide equivalent) compound selectively targets said active agent(or agent capable of becoming an active agent) to apoptotic cells. 2.The method according to claim 1 wherein the system comprises anarsenoxide (or arsenoxide equivalent) compound cleavably linked to anactive agent or an agent capable of becoming an active agent.
 3. Themethod according to claim 1, wherein the system comprises: a firstcomponent comprising an arsenoxide (or arsenoxide equivalent) compoundinked to a first binding member; and a second component, comprising asecond binding member capable of binding to said first binding member,wherein said second binding member is an active agent or an agentcapable of becoming an active agent.
 4. The method according to claim 3,wherein the first binding member is an enzyme and the second bindingmember is a substrate for the enzyme.
 5. The method according to claim4, wherein the substrate for the enzyme is a pro agent which isconverted to an active agent by the enzyme.
 6. The method according toclaim 5, wherein the pro-agent is a prodrug which is converted to anactive drug by the enzyme.
 7. The method according to claim 1, whereinthe system comprises: a first component comprising an arsenoxide (orarsenoxide equivalent) compound linked to a first binding member; and asecond component comprising a second binding member capable of bindingto said first binding member linked to at least one active agent (oragent capable of becoming an active agent). 8-14. (canceled)
 15. Themethod according to claim 7, wherein the second binding member isdirectly or indirectly linked to the active agent.
 16. The methodaccording to claim 15, wherein said indirect linking between the activeagent and the second binding member is by virtue of a further bindinginteraction between the second binding member and at least one furtherbinding member, wherein the further binding member is directly linked tothe active agent.
 17. The method according to claim 7, wherein the firstbinding member is biotin, the second binding member is selected fromavidin and streptavidin, and the second binding member is indirectlylinked to an active agent by virtue of a further binding interactionbetween the avidin or streptavidin and at least one further biotinmoiety which is directly linked to the active agent.
 18. The methodaccording to claim 1, wherein the system comprises: a first componentcomprising an arsenoxide (or arsenoxide equivalent) compound linked to afirst binding member; a second component comprising a second bindingmember capable of binding to said first binding member linked to anenzyme; and a substrate for said enzyme.
 19. The method according toclaim 18, wherein the substrate for said enzyme is a pro agent which isconverted to an active agent by the enzyme.
 20. The method according toclaim 19, wherein the pro-agent is a prodrug. 21-35. (canceled)
 36. Themethod according to claim 1, wherein the arsenoxide (or arsenoxideequivalent) compound is of the formula (I):A-[(XBX′)_(n)B′-Y]_(p) (I)wherein A comprises at least one pendant group; (XBX′)_(n)B′ comprises asuitable linker group, wherein X is selected from the group consistingof —NR, —S(O)—, —S(O)O—, —S(O)₂—, —S(O)₂O—, —C(O)—, -—C(S)—, —C(O)O—,C(S)O—, —C(S)S—, —P(O)(R₁)—, and —P(O)(R₁)O—, or is absent; B isselected from the group consisting of C₁-C₁₀ alkylene, C₂-C₁₀alkenylene, C₂-C₁₀ alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀cycloalkenylene, C₃-C₁₀ heterocycloalkylene, C₅-C₁₀heterocycloalkenylene, C₆-C₁₂ arylene, heteroarylene and C₂-C₁₀ acyl; X′is selected from the group consisting of —NR—, —O—, —S—, —Se—, —S—S—,S(O)—, —OS(O)—, OS(O)O—, —OS(O)₂, —OS(O)₂O—, —S(O)O—, —S(O)₂—, —S(O)₂O—,—OP(O)(R₁)—, —OP(O)(R₁)O—, —OP(O)(R₁)OP(O)(R₁)O—, —C(O)—, —C(S)—,—C(O)O—, C(S)O—, —C(S)S—, —P(O)(R₁)—, —P(O)(R₁)O—, and

or is absent; wherein E is O, S, Se, NR or N(R)₂ ⁺; B′ is selected fromthe group consisting of C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene, C₂-C₁₀alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀ cycloalkenylene, C₃-C₁₀heterocycloalkylene, C₅-C₁₀ heterocycloalkenylene, C₆-C₁₂ arylene, andheteroarylene or is absent; and wherein each R is independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,OR₂ and C₂-C₁₀ acyl; R′ is the same as R or two R′ may be taken togetherwith the nitrogen atoms to which they are attached to form a 5 or6-membered saturated or unsaturated heterocyclic ring; each R₁ isindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl, halo, OR₂ and N(R)₂; each R₂ is independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl and—C(O)R₅; each R₅ is independently selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₁₀ alkoxy, C3-C₁₀alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀ cycloalkyloxy, C₅-C₁₀cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy, C₅-C₁₀heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀ alkylthio,C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀ cycloalkylthio, C₅-C₁₀cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio, C₅-C₁₀heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, OH, SH andN(R)₂; wherein for each instance that B and/or B′ is arylene, thesubstituents directly attached to the respective arylene rings(including arsenoxide or arsenoxide equivalent) may be in a para-, meta-or ortho- relationship; and wherein each alkylene, alkenylene,alkynylene, cycloalkylene, cycloalkenylene, heterocycloalkylene,heterocycloalkenylene, arylene, heteroarylene and acyl may beindependently substituted with hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,cyano, cyanate, isocyanate, OR_(2a), SR₆, nitro, arsenoxide, —S(O)R₃,—OS(O)R₃, —S(O)₂R₃, —OS(O)₂R₃, —P(O)R₄R₄, —OP(O)R₄R₄, —N(R″)₂,—NRC(O)(CH₂)_(m)Q, —C(O)R₅;

wherein R, R₁ and R₅ are as defined above; and R_(2a) is selected fromthe group consisting of hydrogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₆-C₁₂ aryl, —S(O)R₃,—S(O)₂R₃, —P(O)(R₄)₂, N(R)₂ and —C(O)R₅; each R₃ is independentlyselected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,C₁-C₁₀ alkoxy, C₃-C₁₀ alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀cycloalkyloxy, C₅-C₁₀ cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy,C₅-C₁₀ heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio,C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio andN(R)₂; each R₄ is independently selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₁₀ alkoxy, C₃-C₁₀alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀ cycloalkyloxy, C₅-C₁₀cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy, C₅-C₁₀heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀ alkylthio,C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀ cycloalkylthio, C₅-C₁₀cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio, C₅-C₁₀heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, halo and N(R)₂;R₆ is selected from the group consisting of C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio,C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, —S(O)R₃,—S(O)₂R₃ and —C(O)R₅, R″ is the same as R or two R″ taken together withthe N atom to which they are attached may form a saturated, unsaturatedor aromatic heterocyclic ring system; Q is selected from halogen and—OS(O)₂Q₁; wherein Q₁ is selected from C₁-C₄ alkyl, C₁-C₄perfluoroalkyl, phenyl, p-methylphenyl; and m is 1 to 5; n is an integerfrom 0 to 20; Y comprises at least one arsenoxide or arsenoxideequivalent; and p is an integer from 1 to
 10. 37. (canceled)
 38. Themethod according to claim 36, wherein A is selected from the groupconsisting of natural, unnatural and synthetic amino acids, hydrophilicamines, peptides, polypeptides, oligosaccharides, and thiol containingproteins, or a combination thereof.
 39. The method according to claim38, wherein A is selected from the group consisting of glutathione,glucosamine, cysteinylglycine, cysteic acid, aspartic acid, glutamicacid, lysine, and arginine, and wherein the sulfur atom of each sulfurcontaining compound is optionally oxidised to form a sulfoxide orsulfone.
 40. The method according to claim 36, wherein A is glutathione.41-43. (canceled)
 44. The method according to claim 43, wherein thecompound is 4-(N-(S-glutathionylacetyl)amino)-phenylarsenoxide, (GSAO),according to Formula IV:


45. The method according to claim 1, wherein said method comprises thesteps of: (a) administering a first component of the system; (b)optionally waiting for a period of time; and (c) administering a secondcomponent of the system.
 46. The method according to claim 1, whereinsaid method comprises the steps of: (a) administering a first componentof the system; (b) optionally waiting for a period of time; (c)administering a second component of said system; (d) optionally waitingfor a further period of time; and (e) administering a further bindingmember linked to an active agent (or agent capable of becoming an activeagent).
 47. (canceled)
 48. A system for selectively targeting an activeagent (or agent capable of becoming an active agent) to apoptotic cells,said system comprising a first component comprising an arsenoxide (orarsenoxide equivalent) compound linked to an active agent or agentcapable of becoming an active agent.
 49. A system for selectivelytargeting an active agent (or agent capable of becoming an active agent)to apoptotic cells, said system comprising: a first component comprisingan arsenoxide (or arsenoxide equivalent) compound linked to a firstbinding member; and a second component comprising a second bindingmember capable of binding to said first binding member, wherein saidsecond binding member is an active agent or an agent capable of becomingan active agent.
 50. The system according to claim 49, wherein the firstbinding member is an enzyme and the second binding member is a substratefor the enzyme.
 51. The system according to claim 50, wherein thesubstrate for the enzyme is a pro-agent which is converted to an activeagent by the enzyme.
 52. The system according to claim 51, wherein thepro-agent is a prodrug which is converted to an active drug by theenzyme.
 53. A system for selectively targeting an active agent (or anagent capable of becoming an active agent) to apoptotic cells, saidsystem comprising a first component comprising an arsenoxide (orarsenoxide equivalent) compound linked to a first binding member; and asecond component comprising a second binding member capable of bindingto said first binding member linked to at least one active agent (oragent capable of becoming an active agent).
 54. The system according toclaim 50, wherein the second binding member is directly linked to theagent.
 55. The system according to claim 50, wherein the second bindingmember is indirectly linked to the agent.
 56. The system according toclaim 55, wherein indirect linking between the agent and the secondbinding member is by virtue of a further binding interaction between thesecond binding member and at least one further binding member, whereinthe further binding member is directly linked to the agent. 57-64.(canceled)
 65. A system comprising: a first component comprising anarsenoxide (or arsenoxide equivalent) compound linked to a first bindingmember; a second component comprising a second binding member capable ofbinding to said first binding member linked to an enzyme; and asubstrate for said enzyme.
 66. The system according to claim 65, whereinthe substrate for said enzyme is a pro-agent which is converted to anactive agent by the enzyme.
 67. The system according to claim 66,wherein the pro-agent is a prodrug. 68-84. (canceled)
 85. The systemaccording to claim 48, wherein the arsenoxide (or arsenoxide equivalent)compound is of the formula (I): A-[(XBX′)_(n)B′-Y]_(p) (I) wherein Acomprises at least one pendant group; (XBX′)_(n)B′ comprises a suitablelinker group, wherein X is selected from the group consisting of —NR,—S(O)—, —S(O)O—, —S(O)₂—, —S(O)₂O—, —C(O)—, -—C(S)—, —C(O)O—, C(S)O—,—C(S)S—, —P(O)(R₁)—, and —P(O)(R₁)O—, or is absent; B is selected fromthe group consisting of C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene, C₂-C₁₀alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀ cycloalkenylene, C₃-C₁₀heterocycloalkylene, C₅-C₁₀ heterocycloalkenylene, C₆-C₁₂ arylene,heteroarylene and C₂-C₁₀ acyl; X′ is selected from the group consistingof —NR—, —O—, —S—, —Se—, —S—S—, S(O)—, —OS(O)—, OS(O)O—, —OS(O)₂,—OS(O)₂O—, —S(O)O—, —S(O)₂—, —S(O)₂O—, —OP(O)(R₁)—, —OP(O)(R₁)O—,—OP(O)(R₁)OP(O)(R₁)O—, —C(O)—, —C(S)—, —C(O)O—, C(S)O—, —C(S)S—,—P(O)(R₁)—, —P(O)(R₁)O—, and

or is absent; wherein E is O, S, Se, NR or N(R)₂ ⁺; B′ is selected fromthe group consisting of C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene, C₂-C₁₀alkynylene, C₃-C₁₀ cycloalkylene, C₅-C₁₀ cycloalkenylene, C₃-C₁₀heterocycloalkylene, C₅-C₁₀ heterocycloalkenylene, C₆-C₁₂ arylene, andheteroarylene or is absent; and wherein each R is independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,OR₂ and C₂-C₁₀ acyl; R′ is the same as R or two R′ may be taken togetherwith the nitrogen atoms to which they are attached to form a 5 or6-membered saturated or unsaturated heterocyclic ring; each R₁ isindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂aryl, heteroaryl, halo, OR₂ and N(R)₂; each R₂ is independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl and—C(O)R₅; each R₅ is independently selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₁₀ alkoxy, C3-C₁₀alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀ cycloalkyloxy, C₅-C₁₀cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy, C₅-C₁₀heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀ alkylthio,C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀ cycloalkylthio, C₅-C₁₀cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio, C₅-C₁₀heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, OH, SH andN(R)₂; wherein for each instance that B and/or B′ is arylene, thesubstituents directly attached to the respective arylene rings(including arsenoxide or arsenoxide equivalent) may be in a para-, meta-or ortho- relationship; and wherein each alkylene, alkenylene,alkynylene, cycloalkylene, cycloalkenylene, heterocycloalkylene,heterocycloalkenylene, arylene, heteroarylene and acyl may beindependently substituted with hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,cyano, cyanate, isocyanate, OR_(2a), SR₆, nitro, arsenoxide, —S(O)R₃,—OS(O)R₃, —S(O)₂R₃, —OS(O)₂R₃, —P(O)R₄R₄, —OP(O)R₄R₄, —N(R″)₂,—NRC(O)(CH₂)_(m)Q, —C(O)R₅;

wherein R, R₁ and R₅ are as defined above; and R_(2a) is selected fromthe group consisting of hydrogen, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₆-C₁₂ aryl, —S(O)R₃,—S(O)₂R₃, —P(O)(R₄)₂, N(R)₂ and —C(O)R₅; each R₃ is independentlyselected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,C₁-C₁₀ alkoxy, C₃-C₁₀ alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀cycloalkyloxy, C₅-C₁₀ cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy,C₅-C₁₀ heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio,C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio andN(R)₂; each R₄ is independently selected from the group consisting ofhydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀ heterocycloalkyl, C₅-C₁₀heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₁₀ alkoxy, C₃-C₁₀alkenyloxy, C₃-C₁₀ alkynyloxy, C₃-C₁₀ cycloalkyloxy, C₅-C₁₀cycloalkenyloxy, C₃-C₁₀ heterocycloalkyloxy, C₅-C₁₀heterocycloalkenyloxy, C₆-C₁₂ aryloxy, heteroaryloxy, C₁-C₁₀ alkylthio,C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀ cycloalkylthio, C₅-C₁₀cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio, C₅-C₁₀heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, halo and N(R)₂;R₆ is selected from the group consisting of C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₅-C₁₀ cycloalkenyl, C₃-C₁₀heterocycloalkyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₁₂ aryl, heteroaryl,C₁-C₁₀ alkylthio, C₃-C₁₀ alkenylthio, C₃-C₁₀ alkynylthio, C₃-C₁₀cycloalkylthio, C₅-C₁₀ cycloalkenylthio, C₃-C₁₀ heterocycloalkylthio,C₅-C₁₀ heterocycloalkenylthio, C₆-C₁₂ arylthio, heteroarylthio, —S(O)R₃,—S(O)₂R₃ and —C(O)R₅, R″ is the same as R or two R″ taken together withthe N atom to which they are attached may form a saturated, unsaturatedor aromatic heterocyclic ring system; Q is selected from halogen and—OS(O)₂Q₁; wherein Q₁ is selected from C₁-C₄ alkyl, C₁-C₄perfluoroalkyl, phenyl, p-methylphenyl; and m is 1 to 5; n is an integerfrom 0 to 20; Y comprises at least one arsenoxide or arsenoxideequivalent; and p is an integer from 1 to
 10. 86. (canceled)
 87. Thesystem according to claim 85, wherein A is selected from the groupconsisting of natural, unnatural and synthetic amino acids, hydrophilicamines, peptides, polypeptides, oligosaccharides, and thiol containingproteins, or a combination thereof.
 88. The system according to claim87, wherein A is selected from the group consisting of glutathione,glucosamine, cysteinylglycine, cysteic acid, aspartic acid, glutamicacid, lysine, and arginine, and wherein the sulfur atom of each sulfurcontaining compound are optionally oxidised to form a sulfoxide orsulfone.
 89. The system according to claim 85, wherein A is glutathione.90-92. (canceled)
 93. The system according to claim 85, wherein thecompound is 4-(N-(S-glutathionylacetyl)amino)-phenylarsenoxide, (GSAO),according to Formula IV:


94. A method of treatment and/or prophylaxis of a disease in avertebrate in need of said treatment and/or prophylaxis, said methodcomprising administering to said vertebrate a therapeutically effectiveamount of a system comprising an arsenoxide (or arsenoxide equivalent)compound and a therapeutic agent, wherein said arsenoxide (or arsenoxideequivalent) compound selectively targets said agent to apoptotic cells.95. The method according to claim 94, wherein the disease is selectedfrom the group consisting of angiogenesis-dependent diseases, cellularproliferative diseases, inflammatory disorders, auto-immune diseases,blood vessel diseases, thrombosis, cancer, neurodegenerative diseases,myelodysplastic syndromes and ischaemia/reperfusion injury.
 96. A methodof detecting and/or imaging apoptotic cells in a vertebrate, said methodcomprising administering to said vertebrate a biologically effectiveamount of a system comprising an arsenoxide (or arsenoxide equivalent)compound and a diagnostic agent, wherein said arsenoxide (or arsenoxideequivalent) compound selectively targets said agent to apoptotic cells,and detecting said diagnostic agent.
 97. A therapeutic and/or diagnostickit for selectively targeting an active agent (or agent capable ofbecoming an active agent) to apoptotic cells, said kit comprising afirst component comprising an arsenoxide (or arsenoxide equivalent)compound linked to a first binding member, and a second componentcomprising a second binding member capable of binding to said firstbinding member, wherein said second binding member is an active agent oragent capable of becoming an active agent, optionally together with atherapeutically and/or diagnostically acceptable carrier and/or diluent.98. A therapeutic and/or diagnostic kit for selectively targeting anactive agent (or agent capable of becoming an active agent) to apoptoticcells, said kit comprising a first component comprising an arsenoxide(or arsenoxide equivalent) compound linked to a first binding member;and a second component comprising a second binding member capable ofbinding to said first binding member linked to an active agent (or agentcapable of becoming an active agent), optionally together with atherapeutically and/or diagnostically acceptable carrier and/or diluent.99. A therapeutic and/or diagnostic kit for selectively targeting anactive agent (or agent capable of becoming an active agent) to apoptoticcells, said kit comprising a first component comprising an arsenoxide(or arsenoxide equivalent) compound linked to a first binding member; asecond component comprising a second binding member capable of bindingto said first binding member; and a third component comprising a thirdbinding member capable of binding to said second binding member linkedto an active agent (or agent capable of becoming an active agent),optionally together with a therapeutically and/or diagnosticallyacceptable carrier and/or diluent.
 100. (canceled)
 101. A system forselectively targeting an active agent (or agent capable of becoming anactive agent) to apoptotic cells, said system comprising: a firstcomponent comprising an arsenoxide (or arsenoxide equivalent) compoundlinked to a first binding member; a second component comprising a secondbinding member capable of binding to said first binding member; and athird component comprising a third binding member capable of binding tosaid second binding member, wherein said third binding member is linkedto an active agent (or agent capable of becoming an active agent).